Microbial Polymers: Applications and Ecological Perspectives 9811600449, 9789811600449

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Anukool Vaishnav Devendra Kumar Choudhary  Editors

Microbial Polymers

Applications and Ecological Perspectives

Microbial Polymers

Anukool Vaishnav • Devendra Kumar Choudhary Editors

Microbial Polymers Applications and Ecological Perspectives

Editors Anukool Vaishnav Department of Biotechnology GLA University Mathura, India

Devendra Kumar Choudhary Amity Institute of Microbial Technology Amity University Noida, India

ISBN 978-981-16-0044-9 ISBN 978-981-16-0045-6 https://doi.org/10.1007/978-981-16-0045-6

(eBook)

# The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

The trend in polymer sciences during early twentieth century was using synthetic plastic, oil-derived plastics, and their products in burgeoning chemical industry. However, discarded synthetic polymers contribute to the pollution of the environment, and the bulk ends up on beaches, in the oceans, or clogging landfill sites. Recently, there has been an increased awareness of and a sense of responsibility for pollution. This has led to a determined search for biodegradable alternatives to synthetic polymers. Polymers of natural origin (biopolymers) have become increasingly important for both scientific and industrial sectors due to their renewable nature, unique properties, and wide availability. Increased bio-based polymer production and application has been positioned as one of the most promising ways to meet the sustainable development goal of replacing traditional petroleum polymers in several industrial sectors. In this context, microbe-based polymers, including exopolysaccharides, chitin, polyhydroxybutyrates (PHB), polyhydroxyalkanoates (PHAs), fungal mycelium, bacterial pigments and biofilms, have attracted a significant amount of interest recently, especially for industrial, medical, packaging, and agricultural sectors. Microorganisms efficiently convert different carbon sources into a diverse range of polymers, i.e., polysaccharides, polyesters, polyamides, and inorganic polyanhydrides with varying chemical and material properties. Most of the polymers secreted by microbes are extracellular, hence easy to extract them through fermentation. Some of the polymers from different organisms have same functions, whereas other polymers are specific for certain microbial species. Recent discoveries in molecular tools and our understanding about microbial synthesis of polymers have gained interest in rational engineering in microbes towards production of desired polymers with specific high value application at viable economic cost. The fungal mycelium has attracted increasing interests recently as a new form of low energy bio-fabrication and waste upcycling. Fungal mycelium binds organic matter tightly in their hyphal filamentous network that can be exploited to produce packaging and composite materials from agricultural and industrial waste. In agriculture, some microbial polymers such as PHAs are used as a carrier material for controlled and targeted pesticides delivery. Cell encapsulation method is also used for microbial inoculants delivery into soil. In this method, microbial cells such as plant growth-promoting rhizobacteria (PGPR) and arbuscular mycorrhizae fungi (AMF) encapsulate in gel matrix. More than 80% of v

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Preface

bio-encapsulation processes are carried out using various types of alginates. Alginates, linear polymers of b (1,4)-D-mannuronic acid and a(1,4)-L-guluronic acid monomers, are produced by brown algae. and their composition varies depending on the algal source. Likewise, several microbial polymers are already produced commercially including Chitosan, Curdlan, Beta-Glucan, Xanthan Gum, Agar Gum, Gallan Gum, etc. for different industrial purposes. Therefore, significant attention has been paid on the improvement of those sustainable polymers, overall their production, properties, and performance. Hence, in this book, editors compiled researches carried out on microbe-based polymers and their application in diverse sectors including agriculture, food, and medicine. Mathura, India Noida, India

Anukool Vaishnav Devendra Kumar Choudhary

Contents

Part I 1

Diversity of Microbial Polymers

The Production and Applications of Microbial-Derived Polyhydroxybutyrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S. I. Magagula, M. Mohapi, J. S. Sefadi, and M. J. Mochane

2

Fungal Exopolysaccharides: Types, Production and Application . . . Ashim Debnath, Bimal Das, Maimom Soniya Devi, and Ratul Moni Ram

3

Isolation and Purification of Microbial Exopolysaccharides and Their Industrial Application . . . . . . . . . . . . . . . . . . . . . . . . . . . Veena S. More, Allwin Ebinesar, A. Prakruthi, P. Praveen, Aneesa Fasim, Archana Rao, Farhan Zameer, K. S. Anantharaju, and Sunil More

3 45

69

4

A Review on Properties and Applications of Xanthan Gum . . . . . . Surabhi Chaturvedi, Sanchita Kulshrestha, Khushboo Bhardwaj, and Rekha Jangir

87

5

Biosynthesis and Characterization of Poly-(3)-hydroxyalkanoic Acid by Bacillus megaterium SF4 Using Different Carbohydrates . . 109 Temitope O. Fadipe, Nazia Jamil, and Adekunle K. Lawal

6

Mushroom Mycelia-Based Material: An Environmental Friendly Alternative to Synthetic Packaging . . . . . . . . . . . . . . . . . . . . . . . . . 131 Abhik Mojumdar, Himadri Tanaya Behera, and Lopamudra Ray

7

An Overview of Microbial Derived Polyhydroxybutyrate (PHB): Production and Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . 143 Monika Sharma and Harish Kumar Dhingra

8

Insight of Biopolymers and Applications of Polyhydroxyalkanoates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 Rishabh Agrahari, Gargi Sarraf, Naveen Chandra Joshi, Swati Mohapatra, and Ajit Varma

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Contents

Microbial Pigments and Their Application . . . . . . . . . . . . . . . . . . . 193 Selvaraju Vishnupriya, Sundaresan Bhavaniramya, Dharmar Baskaran, and Arulselvam Karthiayani

Part II

Microbial Polymers in Agriculture

10

Extracellular Polymeric Substances from Agriculturally Important Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 Valeria Valenzuela Ruiz, Roel Alejandro Chávez Luzania, Fannie Isela Parra Cota, Gustavo Santoyo, and Sergio de los Santos Villalobos

11

Significance of Bacterial Polyhydroxyalkanoates in Rhizosphere . . . 235 Sundaresan Bhavaniramya, Selvaraju Vishnupriya, and Dharmar Baskaran

12

Role of Microbial Biofilms in Agriculture: Perspectives on Plant and Soil Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 Anupam Gogoi, Mandeep Poudel, Jagajjit Sahu, and Geetanjali Baruah

13

Biological Soil Crusts to Keep Soil Alive, Rehabilitate Degraded Soil, and Develop Soil Habitats . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 Atoosa Gholamhosseinian, Adel Sepehr, Behnam Asgari Lajayer, Nasser Delangiz, and Tess Astatkie

14

Fungal Chitosan: The Importance and Beneficiation of this Biopolymer in Industrial and Agricultural Process . . . . . . . . . . . . . 311 Allwin Ebinesar, Veena S. More, D. L. Ramya, G. R. Amrutha, and Sunil S. More

15

Role of Microbial Extracellular Polymeric Substances in Soil Fertility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 Alok Bharadwaj

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Microbes Derived Exopolysaccharides Play Role in Salt Stress Alleviation in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355 Purnima Singh, Vibha Pandey, and Prerana Parihar

Part III

Microbial Polymers in Industrial Sectors

17

Microbial Exopolysaccharides: Structure and Therapeutic Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 Hiran Kanti Santra and Debdulal Banerjee

18

Microbial Biopolymers: Pharmaceutical, Medical, and Biotechnological Applications . . . . . . . . . . . . . . . . . . . . . . . . . . 421 Rohit Godbole, Asha Goutam, and Aniket Mali

Contents

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Mycobacterium Biofilms Synthesis, Ultrastructure, and Their Perspectives in Drug Tolerance, Environment, and Medicine . . . . . 465 Kundan Kumar Chaubey, Mohd. Abdullah, Saurabh Gupta, Manthena Navabharath, and Shoor Vir Singh

20

A Comprehensive Review on Different Microbial-Derived Pigments and Their Multipurpose Activities . . . . . . . . . . . . . . . . . . . . . . . . . . 479 Archana S. Rao, Sidhartha Pratim Deka, Sunil S. More, Ajay Nair, Veena S. More, and K. S. Ananthjaraju

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Microbial Polysaccharides with Potential Industrial Applications: Diversity, Synthesis, and Their Applications . . . . . . . . . . . . . . . . . . 521 Himadri Tanaya Behera, Abhik Mojumdar, Chiranjib Mohapatra, and Lopamudra Ray

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Eco-friendly Microbial Biopolymers: Recent Development, Biodegradation, and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . 547 Chandrabose Selvaraj and Sanjeev Kumar Singh

Part IV

Advances in Microbial Polymers

23

Microbial Biopolymers as an Alternative Construction Binder . . . . 581 Gye-Chun Cho, Ilhan Chang, and Jooyoung Im

24

Genetic Engineering Approaches for High-End Application of Biopolymers: Advances and Future Prospects . . . . . . . . . . . . . . . 619 Saurabh Gupta, Kundan Kumar Chaubey, Vishal Khandelwal, Tarubala Sharma, and Shoor Vir Singh

25

Microbial Pigments: Secondary Metabolites with Multifaceted Roles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 631 Himadri Tanaya Behera, Abhik Mojumdar, Suchismita Nivedita, and Lopamudra Ray

26

Bio-fermentative Production of Xanthan Gum Biopolymer and Its Application in Petroleum Sector . . . . . . . . . . . . . . . . . . . . . 655 Nanthakumar Kuppanan, Paul Jeyaseelan, Shishram Chahar, Subhasis Das, Veeranna Channashettar, and Banwari Lal

27

A Comparative Study on Biodegradable Packaging Materials: Current Status and Future Prospects . . . . . . . . . . . . . . . . . . . . . . . 675 Archana S. Rao, Ajay Nair, Sunil S. More, Arpita Roy, Veena S. More, and K. S. Anantharaju

28

Environmental Implications of Microbial Bioplastics for a Sustainable Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 695 A. Mbotho, S. I. Magagula, K. M. Moloantoa, J. S. Sefadi, and M. J. Mochane

About the Editors

Dr. Anukool Vaishnav is working as an Assistant professor in Department of Biotechnology, GLA University, Mathura. He operated SERB-National Postdoctoral Fellowship (NPDF) project as a Principal Investigator at Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, India. He has 7 years of research experience in Agriculture Microbiology. His research is mainly focuses on microbial mediated plant protection against biotic and abiotic stress, characterization of signaling molecules and secondary metabolites (soluble and VOCs) in plant holobionts. As an active researcher, he has published more than 20 research and review articles along with 13 book chapters for reputed journals and edited books. He has filed 9 Indian patents in association with his research group. He is an editorial member of Current Genomics Journal published by Bentham Science. He has been awarded with Young Scientist Award from reputed societies. Dr. Devendra Kumar Choudhary is selected member-National Academy of Sciences, India (NASI) and shows his presence at Amity University Uttar Pradesh, Noida. Recently he was associated with PDM and worked as Professor (Ad hoc) for three months. He is active researcher and operated major projects with worth amount 10 million sponsored by DBT, DST and SERB, New Delhi, India, as principal investigator and co-investigator. He has involved and contributed in NAAS policy paper number 36 in the year 2006. He has published more than 80 research/review articles along with several book chapters for reputed journals and edited books (Springer, Elsevier and Taylor Francis/Wiley-Blackwell). He has supervised 08 research scholars for their doctoral programme as supervisor and co-supervisor. In addition, he is recipient of Indian National Science Academy (INSA) visiting and summer research fellowship-2014 and his team received DR. RS Rana Memorial best research award 2013 sponsored by Association of Microbiologists of India. His team filed 3 patents and submitted bacterial strains to public domain wherein incurred two accession numbers from MTCC, IMTECH for submitted bacterial cultures (MTCC, 12057 & 12058) along with one MCC no 2607.

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Part I Diversity of Microbial Polymers

1

The Production and Applications of Microbial-Derived Polyhydroxybutyrates S. I. Magagula, M. Mohapi, J. S. Sefadi, and M. J. Mochane

Abstract

For the last few decades, PHBs have been used to address issues around environmental pollution. This is due to their biodegradable nature when compared with conventional plastics. Moreover, their 100% biodegradability combined with biocompatibility has secured a place for PHBs in many industrial applications such as biomedical, packaging, tissue engineering and drug delivery. However, properties such as brittleness, thermal instability and narrow processing window limit the application of PHBs in many industrial applications. Therefore, in order to overcome this setback, PHBs are usually blended with other polymers, fibres or additives to obtain PHB-based composites with optimum properties. PHBs are a group of PHAs that are synthesized and stored by many microorganisms as a source of carbon and energy. With the growing environmental concerns, there is an increasing demand for the production of PHB-based materials on a larger scale. However, the major problem facing by large-scale production of PHB right now is the high cost of production. In spite of the many attempts to reduce the cost of production, there is still a need for more ways of significantly reducing the production cost. Some of the ways are briefly mentioned in this chapter. This chapter is aimed at providing a detailed discussion on the production and applications of microbial PHBs.

S. I. Magagula · M. Mohapi · M. J. Mochane (*) Department of Life Sciences, Central University of Technology, Bloemfontein Campus, Bloemfontein, South Africa e-mail: [email protected] J. S. Sefadi (*) School of Natural and Applied Sciences, Sol Plaatje University, Kimberly, South Africa e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_1

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S. I. Magagula et al.

Keywords

Polyhydroxybutyrate · Bacteria · Cyanobacteria · Biodegradable plastic

1.1

Introduction

Polyhydroxybutyrates (PHBs) are biopolymers that are synthesized and accumulated by a wide range of microorganisms as energy reserves, especially when the microbes are grown under stressful conditions (Galia 2010; Getachew and Woldesenbet 2016; Ramadas et al. 2009). PHBs are produced via bacterial fermentation processes where different strains of bacteria are used to synthesize and accumulate PHBs in the form of intracellular granules (Ramadas et al. 2009). The bacteria is usually cultured under limited conditions where one of the nutrients like phosphorus, nitrogen, oxygen or sulphur is absent and the presence of carbon is in abundance (Sudesh et al. 2000; Kessler and Witholt 2001). PHBs form a major part of a group of microbial-derived, lipid-like, water-insoluble polyester molecules called polyhydroxyalkanoates (PHAs) (Raza et al. 2019). PHAs are the major building blocks of a group of biopolymers called biodegradable plastics or biopolymers (Carpine et al. 2020). Figure 1.1 shows a classification of the different types of biodegradable plastics. PHBs have processing properties that are similar to those of synthetic thermoplastics such as polypropylene (Raza et al. 2019; Faria and Martins-Franchetti 2010). They are also biocompatible, hydrophobic and 100% biodegradable (Bugnicourt et al. 2014; Lasprilla et al. 2012). Due to their 100% biodegradability, thermoplastic processability and biocompatibility, PHBs have been used to replace synthetic thermoplastics in several industrial applications such as in the packaging industry (Mousavioun et al. 2012), agricultural industry (Alejandra et al. 2012) as well as in the biomedical (Ke et al. 2017; Ho et al. 2014) and pharmaceutical fields (Villanova et al. 2010). Figure 1.2 shows the general structures of biodegradable and non-biodegradable plastics. Due to their non-degradable nature, synthetic thermoplastics have caused some irreparable damages to the environment (Sharma et al. 2007). Most of the synthetic thermoplastics accumulating in the environment as plastic waste end up in our oceans (Anderson et al. 2018; Maes et al. 2017; Do Sul and Costa 2014), our freshwater (Eerkes-Medrano et al. 2015; Jambeck et al. 2015; Mani et al. 2015), our air, our soil and food (Carpine et al. 2020). This causes devastating damages to the ecosystem. For example, most of the synthetic thermoplastics that end up as micro plastics in the ocean are ingested by aquatic creatures that depend on the ocean to survive such as fish (Anderson et al. 2018; Maes et al. 2017). The micro plastics have been discovered in the digestive systems of many aquatic species (Rummel et al. 2016; Van Cauwenberghe and Janssen 2014). So far, clarity is required on how the ingested plastics affect the organisms (Mintenig et al. 2019). However, so far, it has been reported that ingested micro plastics can induce gut inflammations, but their transportation to other organs has not been reported (Bouwmeester et al. 2015).

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The Production and Applications of Microbial-Derived Polyhydroxybutyrates

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Fig. 1.1 Classification of the different types of biodegradable plastics

Micro plastics have been found in seafood (Tanaka and Takada 2016), salt (Iñiguez et al. 2017), honey (Liebezeit and Liebezeit 2015) and tap water (Kosuth et al. 2017). Furthermore, micro plastic particles have also been found in bottled drinking water, and it was suspected that the packaging might also be a contributor to the contamination (Schymanski et al. 2018). This could cause problems for the human health and ecosystem in the long run. Currently, the global consumption of synthetic thermoplastics is approximately 140 million tons per annum (Carpine et al. 2020). This explains the rate at which plastic waste is accumulating around the world. According to EU officials, plastic waste accounts for about 70% of the trash found in the oceans (Carpine et al. 2020). This makes the plastic waste a global concern. Now, several methods of plastic waste disposal have been used over the years. However, these methods have always been faced with serious challenges. For instance, the degradability of the thermoplastics disposed in landfills has been found to be extremely slow. The incineration of the thermoplastics has been found to generate toxic by-products. For those who have tried recycling, they have found recycling to be time-consuming. In addition to that, recycling has also been found to change the properties of the original plastic material (Costa et al. 2019).

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Fig. 1.2 General structures of biodegradable and non-biodegradable plastics. (Springer open access Urbanek et al. (2018))

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The Production and Applications of Microbial-Derived Polyhydroxybutyrates

7

As a result of their eco-friendliness, PHBs have become solution of choice when it comes to protecting the environment against all environmental hazards that have been caused by the synthetic thermoplastics. PHBs are also the only biodegradable plastics that are 100% biodegradable as compared to other biodegradable plastics. This qualifies them even more as ideal candidates for solving the global environmental concerns (Carpine et al. 2020). Currently, bioplastics, with PHBs included, account for 1% of the plastics produced globally per annum. In spite of this, the bioplastic global market continues to grow at approximately 20–100% per annum. In 2019, the production capacity of bioplastics recorded by European Bioplastics and the Research Institute NovaInstitute was 2.11 million tons, and it is expected to increase to 2.43 million tons by 2024 (Carpine et al. 2020). One of the major drawbacks in the extensive production and commercialization of bioplastics, especially PHBs is their high production cost as compared to synthetic thermoplastics (Carpine et al. 2020). As a result, most research has been geared towards reducing the cost of producing PHBs. This has been achieved by developing new strains of bacteria (Bistué Rovira et al. 2019; Peña et al. 2014a) as well as optimizing fermentation and PHB recovery processes (Peña et al. 2014a; Al-Battashi et al. 2019; Lopez-Arenas et al. 2017; Garcia-Gonzalez et al. 2015; Reddy et al. 2019; Carpine et al. 2020). The increase in the cost of production of PHBs is as a result the high carbon substrate cost as well as the high cost of the processes used to recover the PHB granules from the bacterial cell cytoplasm (Carpine et al. 2020). The high production cost together with properties like thermal instability and poor mechanical performance limit the use of PHBs in many industrial applications (Carpine et al. 2020). Therefore, more effort has been focused on reducing the production cost and improving the mechanical properties as well as the thermal stability of the PHBs (Raza et al. 2019). In order to improve their mechanical and thermal properties for packaging, biomedical and tissue engineering applications, PHBs are incorporated with other materials or biopolymers (Raza et al. 2019). The aim of this chapter is to review the production methods used to produce microbial PHBs and their applications in the biomedical field, packaging industry and in tissue engineering.

1.2

A Brief History of Polyhydroxybutyrates (PHBs)

PHBs were first discovered, isolated and characterized in 1925 by a scientist named Maurice Lemoigne (López-Cortés et al. 2008). Due to their 100% biodegradability, biocompatibility and non-toxicity, PHBs have become some of the most important and commonly used type of PHAs (Bugnicourt et al. 2014; Lasprilla et al. 2012). Now, PHBs are aliphatic polyesters characterized by a linear polymer chain (Raza et al. 2019) (see Fig. 1.3). The linear polymer chain of PHBs is composed of 3-hydroxybutyrate monomers consisting of a short chain length of four to five carbon atoms. Each 3-hydroxybutyrate is composed of a β-hydroxybutyric acid consisting of carboxylic acid (-COOH) and alcoholic (-OH) functional groups

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Fig. 1.3 A typical structure of microbial polyhydroxybutyrates (PHBs) (Carpine et al. 2020). (MDPI Open access)

Table 1.1 Comparison between the physical properties of polyhydroxybutyrates (PHB) and polypropylene (PP) (Verlinden et al. 2007; Hankermeyer and Tjeerdema 1999)

Physical property Crystalline melting point (
C) Crystallinity (%) Molecular weight (Daltons) Glass transition temperature (
C) Density (g/cm3) Young’s modulus (GPa) Tensile strength (MPa) Extension to break (%) UV resistance

PHB 177 60 5  105 2 1.25 3.5 40 6 Good

PP 176 50–70 2  105 10 0.905 1.7 38 400 Poor

(Raza et al. 2019). The PHB polymer chain structure is composed of a carbonyl group that is chromophoric, which gives the PHBs an optically active nature (Raza et al. 2018). However, even though PHBs are optically active, it is important to note that PHBs are stable towards (UV) radiation (Raza et al. 2019) (see Table 1.1). Furthermore, the polymer chain structure of PHBs is also isotactic and semicrystalline with an R-configuration (Raza et al. 2019). PHBs have a molecular mass that varies between 50,000 and 1 million Da. The reason for this wide molecular mass range is because the molecular mass of PHBs depends on production conditions such as the carbon source, bacteria growth conditions and the PHB recovery methods used (Dos Santos et al. 2017; Rai et al. 2011). The molecular weight, melting point, crystallinity and tensile strength of PHBs are comparable with those of polypropylene (PP) as shown by Table 1.1. Therefore, PHBs can be used in the place of polypropylene in many industrial applications (Verlinden et al. 2007). PHBs have a glass transition temperature (Tg) of about 2–5
C (Raza et al. 2019; Carpine et al. 2020) and a melting temperature (Tm) of about 177–180
C (Raza et al. 2019, Carpine et al. 2020), which means they are capable of overcoming the harsh processing conditions associated with injection, blowing and extrusion techniques. PHBs also have a density of about 1.26 g/cm3 as well as a specific gravity of about 1.18 g/cm3 (Raza et al. 2019), which makes them lightweight materials suitable for any industrial application. PHBs have superior barrier properties against oxygen than those of polyethylene terephthalate (PETE) and polypropylene (PP) (Rajan et al. 2018). In addition to that, the water vapour barrier properties as well as the barrier properties against odour and

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The Production and Applications of Microbial-Derived Polyhydroxybutyrates

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Fig. 1.4 General structural formula of polyhydroxybutyrate-co-valerate (PHBV) (Rivera-Briso and Serrano-Aroca 2018). (MDPI Open access)

fat of PHBs are also superior to those of polypropylene (Hankermeyer and Tjeerdema 1999). These excellent barrier properties make PHBs suitable for use in food packaging. Furthermore, PHB has been reported to have a mammalian lethal dose (LD50) of more than 500 mg/kg. Therefore, they have been considered to be non-toxic and suitable for use in the denitrification of drinking water (Hankermeyer and Tjeerdema 1999). Regardless of their superior properties, the applications of PHBs are limited by a few major setbacks. One is that PHBs have poor impact properties (Hankermeyer and Tjeerdema 1999). Their extension at break was about 6% in comparison to the 400% of polypropylene (see Table 1.1). Another major setback is that PHBs decompose 10
C above their melting temperature of 177
C, which makes their processing difficult. However, this problem is usually solved by incorporating long pendant hydroxyl acid monomer unit groups, such as hydroxyvalerate. This results in a polyhydroxybutyrate-co-valerate (PHBV) copolymer with superior strength and flexibility than PHB (Hankermeyer and Tjeerdema 1999). Figure 1.4 shows the general structural formula of PHBV.

1.3

Mechanism for the Biosynthesis of PHB in Microorganisms

PHBs have been reported to be synthesized and accumulated by over 300 microorganisms (Koller et al. 2010; Alarfaj et al. 2015). The most commonly investigated microorganisms for PHB accumulation include genera of Alcaligenes, Azotobacter, Bacillus, Rhizobium, Rhodospirillum and Pseudomonas (Chandani et al. 2014; Jiang et al. 2011; Bhagowati et al. 2015). These microorganisms synthesize and accumulate PHBs as energy and carbon sources as reserves for use when experiencing extremely harsh growth conditions. Therefore, the synthesis of PHBs is usually carried out under unbalanced cellular growth conditions. That is, in the presence of excessive amounts of a suitable carbon source, and a limited supply of essential nutrients such as nitrogen, phosphorus, sulphur or oxygen (Carpine et al. 2020). Table 1.2 shows some of the microorganisms used in the production of PHB, the used feedstock and the type of polymer recovered.

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Table 1.2 Some strains of bacteria used for the synthesis and accumulation of PHB

Type of bacterial strain Aeromonas hydrophila 4AK4 mutant Alcaligenes latus Azotobacter chroococcum H23 Bacillus cereus UW85 Bacillus megaterium ATCC 6748 Bacillus spp. 87I Burkholderia sacchari sp. IPT101 Burkholderia cepacia IPT 048 Caulobacter crescentus DSM 4727 Cupriavidus necator DSM 545 Cupriavidus necator DSM 545 Enterobacter aerogenes 12Bi Escherichia coli mutants Halomonas boliviensis LC1

Legionella pneumophila 74/81 Methylocystis sp. GB 25 DSM 7674 Microlunatus phosphovorus DSM 10555 Mixed microbial culture Pandoraea sp.

Source of carbon Oleic acid, lauric acid

Malt, milk waste, soy waste Wastewater obtained from olive oil mills Glucose

Type of biopolymer (s) produced MCL-PHA

Amount of PHB produced (%) 64

PHB

70

PHB

80

PHB

9

Refs Han et al. (2004) Wong et al. (2004) Pozo et al. (2002) Łabużek and Radecka (2001) Chaijamrus and Udpuay (2008) Thirumala et al. (2010) Gent (2001)

Molasses, corn steep liquor Glucose

PHB

43

PHB

67

Glucose

PHB PHBV

68

Bagasse

PHB

62

Glucose

PHB

18

Corn syrup

PHB

30

Waste glycerol

PHB

62

Wastewater

PHB

43

Xylose

PHB

27

Hydrolysate, starch, maltose, maltohexaose and maltotetraose Nutrient broth

PHB

56

PHB

16

Methane

PHB

51

Glucose, acetate

PHB

30

Lactate

PHB

90

By et al. (2013)

Crude glycerol

PHB

63

de Paula et al. (2017)

Silva et al. (2004) Qi and Rehm (2001) Daneshi et al. (2010) Cavalheiro et al. (2009) Ceyhan and Ozdemir (2011) Nikel et al. (2006) Quillaguaman et al. (2005)

James et al. (1999) Wendlandt et al. (2005) Akar et al. (2006)

(continued)

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Table 1.2 (continued)

Type of bacterial strain Pseudomonas aeruginosa NCIB 40045 Pseudomonas hydrogenovora DSM 1749 Pseudomonas putida CA-3 Pseudomonas fluorescens A2a5 Rhodopseudomonas palustris SP5212 Ralstonia pickettii 61A6

Type of biopolymer (s) produced MCL-PHA

Amount of PHB produced (%) 66

Dairy whey

MCL-PHA

21

Koller et al. (2008)

Petrochemical plastic waste Sugar cane liquor

PHB

30

PHB

70

Acetate

PHB

34

Sugar cane liquor

PHB

10

Goff et al. (2007) Jiang et al. (2008) Mukhopadhyay et al. (2005) Bonatto et al. (2004)

Source of carbon Agro-industrial oily wastes

Refs Fernández et al. (2005)

MCL-PHA medium-chain-length-polyhydroxyalkanoates, PHB polyhydroxybutyrate, PHBV poly (3-hydroxybutyrate-co-3-hydroxyvalerate)

Now, PHB-producing microorganisms synthesize PHBs via three consecutive enzymatic reactions. The first step is the condensation of two acetyl-CoAs to acetoacetyl-CoA by β-ketothiolase (PhaA). The second step involves reducing acetoacetyl-CoA to 3-hydroxybutyril-CoA by an acetoacetyl-CoA reductase (PhaB). Finally, the third step involves the polymerization of 3-hydroxybutyrilCoA monomers to PHB using PHB synthase (PhaC and PhaE) (Sagong et al. 2018) (see Fig. 1.5). Once the PHB is polymerized, it is packed into PHB granules called carbonosomes (see Fig. 1.6). These carbonosomes primarily act as carbon and energy storage materials situated inside the cells of various bacterial strains (Nikel et al., 2006, Asada et al., 1999, Qi and Rehm, 2001, Zhang et al., 2015, Angermayr et al., 2015, Carpine et al., 2020). The carbonosomes consist of PHBs covered by granule-associated proteins (GAPs) (Jendrossek 2009; Pfeiffer and Jendrossek 2012). Now, there are four main types of GAPs reported in literature: PHB depolymerases (PhaZ), PHB synthases (PhaC), phasins (PhaP) and regulators (PhaR or PhaM) (Tirapelle et al. 2013; De Los Angeles Martínez-Martínez et al. 2019). The synthases and depolymerases are responsible for the accumulation and utilization of PHB (Juengert et al. 2018; Mezzolla et al. 2018). Phasins are responsible for coating and stabilizing the PHB in the cytoplasm (Sznajder et al. 2015). In addition to that, phasins are also responsible for regulating the number and size of carbonosomes (Pfeiffer and Jendrossek 2012; Hauf et al. 2015). Furthermore, the regulators (PhaR) are responsible for regulating the synthesis of phasins (Maehara et al. 2002; Pötter et al. 2002) and the localization of the PHB granules (PhaM) (Pfeiffer et al. 2011; Pfeiffer and Jendrossek 2014).

12

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Fig. 1.5 Mechanism for the biosynthesis of PHB (Carpine et al. 2020). (MDPI Open access)

Phasins have been regarded as the most important type of GAPs (Sznajder et al. 2015; De Los Angeles Martínez-Martínez et al. 2019). This is mostly due to the fact that they are able to interact with both PHB depolymerases and PHB polymerases. This depends on the type of microorganism used (Ushimaru et al. 2014; Tian et al. 2005; Handrick et al. 2004a; Handrick et al. 2004b). For example, studies on Aeromonas hydrophila revealed a two-fold increase of the PHB content when only PhaP was overexpressed (Ushimaru et al. 2014). Changes in the composition of the monomers of the poly(3-hydroxybutyrate-co-3-hydrohexanoate) [P(HB-co-HHx)] copolyester were also observed (Tian et al. 2005). Furthermore, studies on Rhodospirillum rubrum revealed that the presence PhP also promoted the degradability of PHB (Handrick et al. 2004b; De Los Angeles Martínez-Martínez et al. 2019). These studies showed that the interaction of the phasins with PHB polymerases and PHB depolymerases has a positive effect on the synthesis and degradability of PHB (De Los Angeles Martínez-Martínez et al. 2019).

1.4

Production of PHB

With the increasing impact of plastics on the environment, there is an increasing need for the production of bioplastics on a larger scale. Currently, the production of bioplastics on a smaller scale in comparison to the production of petroleum-derived plastics (Carpine et al. 2020). From literature, the production of PHB by Brazilian,

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The Production and Applications of Microbial-Derived Polyhydroxybutyrates

13

Fig. 1.6 PHB granules (white inclusions) shown on a transmission electron micrograph of a thin section belonging to A. vinelandii (Peña et al., 2014a). Open Access

British and Chinese bioplastic companies was reported to be 0.1, 0.3 and 10 kilotonnes per year, which is quite low when compared with petroleum-based plastics (Carpine et al. 2020). The question is why? This question will be answered in a moment. Now, due to their 100% biodegradability, eco-friendliness and biocompatibility, PHBs have gained enormous attention in research and numerous commercial applications (Ramadas et al. 2009; Raza et al. 2019; Carpine et al. 2020). The production of PHBs is normally done via the commonly known bacterial fermentation processes (Carpine et al. 2020; Ramadas et al. 2009). The fermentation process can be divided into two phases. In the first phase, all the nutrients (e.g. carbon source, NH3, O2) needed for the growth of the biomass are added into the fermenter in order to raise the biomass concentration to the desired level. In the second phase, the supply of nitrogen is cut off so that only the carbon source and O2 are added into the fermenter. This restricts the biomass from growing so that the excess carbon source is redirected towards PHB production (Rathore 2014). Over the years, the fermentation processes have used a bacterial system called the chemoheterotrophic bacterial system. The chemoheterotrophic bacterial system (as shown in Table 1.2) uses agro-based carbon source materials as feedstock (Carpine et al. 2020). Now, this brings us back to our previous question about why the production of bioplastics such as PHB is done on a small scale. The agrobased carbon source materials used as feedstock for the production of PHB by the chemoheterotrophic bacteria are expensive (Carpine et al. 2020). They occupy 50% of the total cost of production of PHB (Ramadas et al. 2009). Therefore, this limits the production of PHB on a larger scale (Carpine et al. 2020). A lot of work, including the use of waste material as a carbon source (Al-Battashi et al. 2019; Reddy et al. 2019) and improving the fermentation strategy (LopezArenas et al. 2017; Garcia-Gonzalez et al. 2015) is still being conducted for reducing the cost of production of PHBs. Unfortunately, most of the work reported so far is

14

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still based on the chemoheterotrophic bacterial system which uses an expensive feedstock. Therefore, a bacterial system called the photoautotrophic cyanobacteria system has been alternatively used for reducing the cost of production of PHBs (Carpine et al. 2020). The photoautotrophic cyanobacteria system uses CO2 and sunlight as feedstock instead of the expensive carbon source materials. This bacterial system uses different strains of cyanobacteria for the production of PHB. Judging from the previous history of PHB production, no one ever thought that photoautotrophic bacteria like cyanobacteria could ever be used in producing PHB (Carpine et al. 2020). Table 1.3 shows a summary of some of the cyanobacteria strains that are used in producing PHB. Using cheap feedstock such as CO2 and sunlight for the production of PHB by cyanobacteria has attracted a lot of research attention (Carpine et al. 2020). Therefore, different techniques have been developed for the improvement of PHB production by cyanobacteria. These strategies include: (1) designing cyanobacteria growth conditions; (2) genetic engineering of the cyanobacteria for the improvement of the PHB storage as shown in Table 1.4; (3) developing suitable models as tools for identifying the challenges faced by the microbial production of PHB; and (4) facilitating the operation of photobioreactors in order to get a maximum yield (Carpine et al. 2020). The synergy between these strategies is very important for the designing of an optimum PHB production process using cyanobacteria (Carpine et al. 2020). The production of PHB using cyanobacteria has a few challenges as well. The first challenge is that the recovery process for recovering the PHB homopolymer from the bacterial cells is expensive (Carpine et al. 2020). The second challenge is that the chemicals that are used during the recovery process are not environmentally friendly (Carpine et al. 2020). Therefore, a cheaper and eco-friendly recovery process needs to be investigated. Just like their chemoheterotrophic counterparts, cyanobacteria have all the qualities of bacteria (Carpine et al. 2020). However, there is one feature that distinguishes cyanobacteria from their chemoheterotrophic counterparts. That feature is their ability to use CO2 and sunlight energy, instead of the expensive carbon source materials, to convert them into a product of interest such as PHB (Carpine et al. 2020). The use of cyanobacteria for PHB production has two advantages. The first is that cyanobacteria do not use agro-food market products for their feedstock (Kadiyala 2014; Gupta et al. 2013; Wijffels et al. 2013) and the second is that cyanobacteria use captured CO2, thus reducing the pressure of releasing greenhouse gases into the atmosphere (Carpine et al. 2020). Cyanobacteria produce and accumulate PHB when they are exposed to photoautotrophic conditions under a limited supply of nutrients such as nitrate, phosphorus, calcium and magnesium, as well as macro-nutrients such as manganese, ferrous, cobalt, zinc and copper (Balaji et al. 2013; Costa et al. 2018). The accumulated PHB is usually used as carbon and energy storages by the cyanobacteria (Wu et al. 2001). Now, Synechocystis sp. PCC 6803 is one of the most commonly used as well as characterized cyanobacteria strain for the production of PHB. This is because this strain of the cyanobacteria grows very fast. Its doubling time has been reduced to less than 7 h. The other reason for the cyanobacteria strain’s common use is that this

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The Production and Applications of Microbial-Derived Polyhydroxybutyrates

15

Table 1.3 Summary of some of the cyanobacteria strains used for the production of PHB Type of cyanobacterial strain Arthrospira (Spirulina) platensis Anabaena cylindrica 10C Aulosira fertilissima CCC 444 Aulosira fertilissima CCC 444 Gloeothece sp. PCC 6909 Nostoc muscorum Agardh Nostoc muscorum CCAP 1453/9 Scytonema geitleri Bharawaja Synechococcus sp. PCC7942 Synechocystis sp. PCC 6803 Synechocystis sp. PCC 6803 Synechocystis sp. PCC 6803 Spirulina sp. LEB 18 Spirulina platensis UMACC 161

Cyanobacteria growth conditions Photoautotrophic

Propionate Citrate, acetate and K2HPO4

Type of produced biopolymer PHB

P(3HB-co3HV) PHB

Amount of produced biopolymer (%) 6

2 85

77

Refs Campbell et al. (1982) Lama et al. (1996) Samantaray and Mallick (2012) Ceyhan and Ozdemir (2011) Stal et al. (1990) Cavalheiro et al. (2009)

Fructose and valerate

P(3HB-co3HV)

Acetate

PHB

Deficiency of nitrogen, acetate, glucose and valerate CO2

P(3HB-co3HV)

71

PHB

22

Akar et al. (2006)

30 mM acetate

PHB

7

Singh et al. (2019)

Acetate and deficiency of nitrogen CO2; concentration of nitrate is the half of the optimal concentration Deficiency of nitrate; CO2

PHB

26

PHB

8

Nikel et al. (2006) Wendlandt et al. (2005)

PHB

4

Deficiency of phosphate, gas exchange limitation and acetate Sodium bicarbonate, nitrogen and phosphorus deficiency CO2 and acetate

PHB

38

PHB

31

Quillaguaman et al. (2005)

PHB

10

Toh et al. (2008)

9

Qi and Rehm (2001) Goff et al. (2007)

strain of the cyanobacteria does not have specific nutritional requirements (Angermayr et al. 2009). It is flexible.

Type of cyanobacteria strain Synechocystis sp. PCC 6803 mutant strain Synechococcus sp. PCC7942 mutant strain Synechocystis sp. PCC 6803 mutant strain Synechocystis sp. PCC 6803 mutant strain Synechocystis sp. PCC 6803 mutant strain Synechocystis sp. PCC 6803 mutant strain Synechocystis sp. PCC 6803 mutant strain Flask

PBR

Deprived of nitrogen BG11

0.035% CO2, 4 mM acetate 2% CO2

Native PhaAB from the chromosome was overexpressed

Expression of xfpk from B. breve in a double ach and pta knock out

Flask

Deprived of nitrogen

Flask

Deprived of nitrogen

2% CO2

Flask

Deprived of nitrogen

Expression of PHA biosynthetic operon obtained from M. aeruginosa in a plasmid

Expression of native sigE from the chromosome

Expression of PHB synthase from C. necator in a plasmid

Flask

Deprived of nitrogen

Type of reactor Flask

5% CO2, 10 mM acetate 1% CO2, 10 mM acetate 1% CO2

Cyanobacteria growth conditions Deprived of nitrogen

Expression of a PHB synthesizing enzyme obtained from A. eutrophus in a plasmid

Genetic engineering approach Insertion of the agp gene

Source of carbon 0.035% CO2, 5 mM acetate

Table 1.4 Genetic engineering approaches used to improve PHB accumulation in cyanobacteria

12

35

7

1.4

11

25

Amount of PHB accumulated (% dcw) 18.6

Vermaas (2001)

Poirier et al. (1992)

Wijffels et al. (2013)

Blankenship (2010)

Gopi et al. (2014)

Nikel et al. (2006)

Refs Gupta et al. (2013)

16 S. I. Magagula et al.

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1.5

Factors Affecting the PHB Accumulation in Bacteria

1.5.1

Effect of the Bacteria Strain

17

A lot of bacteria strains which have the natural capacity to produce different amounts of PHBs have been used over the years. However, most of these bacteria strains have shown limited industrial applicability during the industrial scale production of PHBs. This is because these bacteria strains produce low yields of PHB with reference to the yields required on an industrial scale. Therefore, to expand PHB production to an industrial scale, more efforts have been geared towards the genetic modification of the bacteria strains. The genetic modification of the bacteria strains was achieved in several ways which include the modification of their metabolism pathways so that they favour the synthesis of PHB, the regulation of the regulatory systems that regulate the synthesis of PHB and expression of the recombinant phb gene (Peña et al. 2014a). The PHB biosynthetic pathways in bacteria contend for the same precursors with the bacteria’s central metabolic pathways. These central metabolic pathways include the tricarboxylic acid (TCA) cycle, degradation of fatty acid (β-oxidation) and biosynthesis of fatty acid. These PHB biosynthetic pathways also contend with many other biosynthetic pathways with which they share common precursors (Peña et al. 2014a). Three examples of PHB synthesis by metabolic pathway modification in A. vinelandii have been reported (Peña et al. 2014a). The first example involved the mutation of the respiratory NDH oxidase to give a new strain (UWD). This strain was able to accumulate PHB in the exponential growth phase without depriving it of nutrients (Page and Knosp 1989). The second instance involved inactivating pyruvate carboxylate, which is the enzyme responsible for catalysing the reactions that replenish the TCA cycle (Segura and Espín 2004). This type of mutation increased the production of PHB three times more than what was produced by the natural PHB-producing strain (A. vinelandii UW136). This was attributed to the blocked flux of acetyl-CoA into the TCA cycle and hence increasing its availability for PHB synthesis. The third example involved the mutation that blocks the synthesis of the alginate exopolysaccharide. This mutation increased the production of PHB five times more than what is produced by the natural species (UTCC9046). This mutation did not only increase the content of PHB per biomass unit produced by the bacterium, but it also increased the bacterium growth rate. As a result, the volumetric polymer production was increased by 10-fold (Segura et al. 2003). A few examples involving PHB synthesis by the modification of the regulatory systems that regulate the synthesis of PHB in A. vinelandii have also been reported. PHB synthesis in A. vinelandii is regulated by two systems (Peña et al. 2014a). The first system is called the nitrogen-related phosphotransferase system, in which the expression of the PHB biosynthetic operon is suppressed by the IIANtr protein in a non-phosphorylated form (Noguez et al. 2008). The second system is called the posttranscriptional regulatory system (RsmZ/Y- A). In this PHB synthesis system, the translation of mRNAs from phbBAC biosynthetic operon and the phbR that codes for

18

S. I. Magagula et al.

the RsmA protein’s transcriptional activator is suppressed by the RsmA protein (Hernandez-Eligio et al. 2012). In order to improve PHB production, the IIANtr and RsmA-negative regulators were first identified. A. vinelandii was used in the production of PHB by inactivating the gene coding in its IIANtr (ptsN) protein. As a result of the mutation, a 77% PHB yield was achieved which was equivalent to 4.1 g/ L in the genetically modified strain and 3.5 g/L in the natural strain. Therefore, this means that the obtained yield was increased by 36% when compared to the original strain depending on the substrate consumed (Peña et al. 2014b). To further increase PHB production, both the IIANtr and RsmA-negative regulators were inactivated. This mutation together with a suitable fermentation process further increased the PHB production up to 27 g/L (García et al. 2014). Another example involving the synthesis of PHB by the modification of the regulatory systems that regulate PHB synthesis in cyanobacterium Synechocystis sp. PCC 6803 was reported. This is also shown in Table 1.4. Here, the overexpression of the sigma factor SigE, which is responsible for the activation of many catabolic genes of sugar and increasing the acetyl-CoA levels, increased PHB production by two- or three-fold compared to the natural strain (Osanai et al. 2013). The successful production of PHB is usually determined by the fast growth rate of the bacterium and an increased cell density consisting of an increased PHB content in the culture media. E. coli, a well-investigated bacterium using several state-of-theart technologies used in genetic engineering has attracted a lot of research attention for its use in the production of PHB.E. coli is a non-producer of PHB. However, in 1988 Slater et al. (Slater et al. 1988) cloned for the first time the genes of the PHB producer strain, C. necator H16 in E. coli. The experiment was a success and it resulted in the production of PHB by E. coli. Since then, a lot of genetic modifications have been performed on E. coli for improving PHB accumulation in these microorganisms. Most of the strategies for genetic modification have been reported by Li et al. (2007) and Wang et al. (2013). Most studies have shown that the use of recombinant E. coli strains increases the cell concentration in cultures up to 200 g dry cell weight (DCW)/L, cell growth rate and PHB content up to 90% of the DCW than in natural species (Ahn et al. 2000; Reddy et al. 2003).

1.5.2

Effect of the Carbon Source Materials

The cost of producing PHB is usually influenced by several factors which include fermentation substrate costs, polymer extraction from the bacteria cell cytoplasm as well as the handling the extraction and fermentation wastes (Chen 2010). However, of all the costs involved in the production of PHBs, the carbon source materials cost is the highest (Peña et al. 2014a). The carbon source materials used in the production of PHB can be categorized into two categories; the agro-based and non-agro materials such as CO2 and sunlight. The agro-based carbon source materials are used by most chemoheterotrophic bacterial systems such as the ones shown in Table 1.2 and the non-agro materials are used by most photoautotrophic bacterial systems such as cyanobacteria as shown by Table 1.3. The use of agro-based carbon

1

The Production and Applications of Microbial-Derived Polyhydroxybutyrates

19

source materials is very expensive because it competes with the agro-food market (Carpine et al. 2020). Therefore, new strategies which use waste materials (Al-Battashi et al. 2019; Reddy et al. 2019), CO2 and sunlight (Carpine et al. 2020) as carbon source materials have been introduced as new ways of reducing the production cost of PHBs. For example, the use of several bacteria strains to convert wastewater (Reddy et al. 2019), paper waste (Al-Battashi et al. 2019), CO2 and sunlight (Carpine et al. 2020) into PHB has been considered a cheaper PHB synthetic route. When a suitable carbon source material is selected, the standard of selection should not be based only on its market price but on its availability and global price as well (Chanprateep 2010). Table 1.2 shows a summary of some of the agro-based carbon sources used in the production of PHB. From the information presented in the table, it is clear that the agro-based carbon source material used as the fermentation substrate has a significant effect on the amount of PHB produced. For example, when corn syrup and waste glycerol were used in the production of PHB using Cupriavidus necator DSM 545 bacteria, 30 and 62% of PHB were accumulated. Contrastingly, Table 1.4 shows a summary of some of the non-agro carbon sources used in the production of PHB. From the table, it was also observed that the non-agro carbon source materials significantly influenced the PHB content accumulated by cyanobacteria. For example, the use of 2% CO2 as well as 0.035% CO2 and 4 mM acetate as carbon source materials in the presence of Synechocystis sp. PCC 6803 mutant strain under nitrogen-deprived conditions, gave PHB yields of 7% and 35%. This was an indication that both agro-based and non-agro carbon source materials had an influence on the amount of PHB accumulated by bacteria.

1.5.3

Effect of the Fermentation Process

Over the last few decades, very few bacteria strains including C. necator and recombinant E. coli have been employed in the industrial scale synthesis of PHB (Khanna and Srivastava 2005). Surprisingly, high PHB content accumulating bacteria strains such as A. vinelandii and A. chrooccum have not been used in the industrial scale production of PHB (Peña et al. 2014a). As a result, more bacteria strains have been added to the list of bacteria strains used in the industrial scale production of PHB. Many fermentation processes which use fed-batch, batch and continuous cultures are used in PHBs production (Peña et al. 2014a; Sirohi et al. 2020). Fermentation processes using batch cultures for PHB production have been the most commonly used as a result of their low cost of operation and flexibility. However, their disadvantage is that batch cultures produce a low PHB yield (Peña et al. 2014a). For example, the production of PHB in batch cultures consisting of C. necator ATCC 17699 and acetic acid as the source of carbon was investigated by Wang and Yu (Wang and Yu 2001). In their research, only a PHB content of 50% (w/w) and productivity rate of 0.046 g/Lh was achieved with a carbon/nitrogen (C/N) ratio of 76 in their batch culture conditions.

20

S. I. Magagula et al.

Fermentation processes involving the use of fed-batch cultures have been used to obtain increased yields of PHB (Kulpreecha et al. 2009). During fed-batch cultivation systems, one or several nutrients are fed into the bioreactor. The obtained products and other constituents of the system are left inside the bioreactor until the fermentation process is done. This allows materials to only flow in and not out. As a result of this, the contents of the bioreactor keep changing with time (Mejía et al. 2010). In order to regulate the carbon source materials concentration during the fed-batch process, a good substrate feeding strategy is required. There are several feeding strategies used which include, pulse feeding, continuous feeding with a defined feeding rate, exponential feeding, using pH control (pH-stat mode) and dissolved oxygen (DO) concentration to control the nutrient feed (DO-stat mode) (Cruz et al. 2019). From previous studies, it has been shown that the feeding strategy used in fed-batch cultures has a significant effect on the amount of PHB accumulated as shown by Table 1.5. Fermentation processes involving the use of a continuous culture system usually produce PHB in two or three stages (Ruan et al. 2003; Rocha et al. 2008). The first stage is for biomass accumulation and the second stage is for PHB production. The continuous culture PHB production process has attracted a lot of commercial interest. This is due to its high PHB productivity rate especially when strains such as R. eutropha characterized by an increased specific rate of growth are used. For example, Du et al. (2001) obtained a maximum PHB yield of 72.1% at a dilution rate of 0.075 h1 when a two-stage continuous culture of R. eutropha was used in the presence of glucose as the carbon source. In another study, Tan et al. (2011) obtained a maximum PHB yield of 65–70% after 14 days when an open unsterile continuous culture of HalomonasTD01 was used in the presence of a nitrogen-deficient glucose salt medium.

1.5.4

Effect of Culture Conditions

The effect of culture conditions such as pH, medium composition and oxygen availability on the molecular weight of PHB has been reported by several authors (Peña et al. 2014a). Table 1.6 shows a summary of the effect of different culture conditions on PHB molecular weight. The PHB molecular weight is an important feature that determines its medical applications. Therefore, the molecular weight of PHB is highly considered during the commercial production of PHB (Peña et al. 2014a). Moreover, properties such as the elastic behaviour and mechanical resistance of the PHB are controlled by its molecular weight. For instance, PHB fibres with a molecular weight of about 3.0  102 kDa are characterized by a tensile strength of about 190 MPa as well as an elongation at break of about 5%. Contrastingly, PHB fibres with a higher molecular weight of about 5.3  103 can experience a seven-fold increase of their tensile strength up to 1320 MPa and an increase of their elongation at break up to 57% (Iwata 2005).

46

1.73 g L1 h1

Sugar cane molasses

12.6  0.8 g L1 day1

27.2  0.5 19.8  1.8

DO-stat

B. megaterium BA-019

84.0  4.5 77.0  5.7 42

4.5  0.2 g L1 day1

21.3  0.9 17.9  0.9

Used cooking oil

C. necator DSM 428

Single pulse feeding (300 mL) with supplements 4 times pulses feeding (each 75 mL) with supplements Exponential feeding

72.6 90.7

Intermittent

41.6

30.5

4.47

n.m.

pH-stat

4.02

n.m.

n.m.

0.08 g L1 h1

1.27 g L1 h1

n.m.

0.09 g L1 h1

0.09 g L1 h1

Corn stover alkaline pretreatment liquor

n.m.

3.54

Intermittant  DO-stat

Cheese whey

1.40  0.04 g L1 h1

31.7  1.4

Methylobacterium sp. ZP24 C. necator

n.m.

Sucrose

E. Coli W

1.87  0.05 g L1 h1

36.0  0.4

n.m.

45.8  3.6 40.1  0.4 36.2  1.2 64

Glucose

17.5  0.01 g L1 h1

47.7  3.1

n.m.

PHB content (% wt) n.m.

E. Coli WΔcscR mutant E. Coli W

PHB productivity n.m.

PHB concentration (g L1) 11.32

Carbon source Feeding strategy Dairy DO-stat mode waste + rice bran + seawater Sucrose pH-stat mode

Bacteria strain B. megaterium SRKP-3

Dry cell weight (DCW) (g L1) n.m.

Table 1.5 Effect of different feeding strategies on the PHB production in fed-batch cultures

(continued)

Kulpreecha et al. (2009) Kanjanachumpol et al. (2013)

Cruz et al. (2019)

Li and Wilkins (2020)

Nath et al. (2008)

Arifin et al. (2011)

Refs Pandian et al. (2010)

1 The Production and Applications of Microbial-Derived Polyhydroxybutyrates 21

82.5 164

Pulses

Pulses

Exponential  coupled to alkali addition monitoring  constant with N2 limitation pH-stat Exponential  pulses

Soybean oil

Glycerol

Glucose

n.m. stands for not mentioned

119.5 37.2

83

Feeding strategy Pulses

Dry cell weight (DCW) (g L1) 75

Carbon source Glycerol

E. coli CGSC 4401 Whey A. vinelandii Sucrose and yeast extract

Bacteria strain C. necator

Table 1.5 (continued)

96.2 27.3

125

51.2

67.2

PHB concentration (g L1) 53

PHB content (% wt) 71 81 62 76.2

80 73.3

PHB productivity 0.92 g L1 h1 2.5 g L1 h1 1.52 g L1 h1 2.03 g L1 h1

2.57 g L1 h1 0.5 g L1 h1

Ahn et al. (2000) García et al. (2014)

Refs Tanadchangsaeng and Yu (2012)) da Cruz Pradella et al. (2012) Cavalheiro et al. (2009) Mozumder et al. (2014)

22 S. I. Magagula et al.

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1.6

23

Extraction of PHB

The production of PHB usually involves the fermentation, extraction and purification of PHB. Now, the commercial PHB production and industrial PHB application is limited by its expensive production cost when compared to petroleum-derived plastics. The high production cost is mostly influenced by the cost of the fermentation process and the extraction/purification technologies. In addition to the fermentation cost, the PHB recovery process also increases the overall production cost quite significantly (Aramvash et al. 2015). In order to reduce the PHB extraction costs, solvents such as sodium hydroxide and sulphuric acid have been used during the extraction process (López-Abelairas et al. 2015). Unfortunately, the use of these solvents has been considered not environmentally friendly. Therefore, an environmentally friendly, more efficient and cheaper PHB extraction method is needed. In order to develop a suitable extraction process, more details on the formation and accumulation of PHB need to be provided. PHB is a compound belonging to a class of polyesters that are produced by several bacteria strains and then stored as a carbon and energy reserve. The PHB is accumulated in the cell cytoplasm of the microorganisms in the form of inclusion bodies or PHB granules. The PHB granules appear as sack-like structures wrapped up by layer made up of a lot of proteins (see Fig. 1.4). The PHB granules have supramolecular complexes-like structures and specific functions which make them to be more than just carbon and energy storage materials (Jendrossek and Pfeiffer 2014). The mechanism for the formation of PHB granules in bacteria is a topic that is currently under intense discussion. In order to understand the mechanisms that lead to the formation of PHB granules, three models were developed. These models were developed from theoretical manipulations and experiments performed on cultures of Ralstonia eutropha. The first model is called the micelle model. This model is based on the assumption that the soluble (cytoplasmic) PHB synthase can only start producing PHB once the substrate (3-hydroxybutyryl-CoA) concentration has increased sufficiently (Gerngross et al. 1993). During the PHB production, the nascent PHB polymer chains form hydrophobic and less soluble aggregates which form micelle structures in the cell cytoplasm. According to the micelle model, PHB granules assume a random distribution within the cell (Jendrossek and Pfeiffer 2014; Stubbe et al. 2005). The second model is called the budding model. According to this model, the PHB synthase is situated in the cytoplasm membrane and the PHB granules that have been newly formed are found in the membrane bilayer (Das et al. 1997). The third model is called the scaffold model. According to this model, it is assumed that the PHB synthase of nascent PHB granules attaches itself on a scaffold molecule that is yet unknown in the cell (Jendrossek and Pfeiffer 2014). This forms a granule-scaffold complex that is attached to the bacteria nucleoid and consists of complexes that initiate PHB granule formation. According to the scaffold model, the subcellular localization of the PHB granules is determined by the localization and nature of the scaffold molecules consisting of the cells accumulating PHB (Jendrossek and Pfeiffer 2014).

Sucrose

Sucrose

A. vinelandii OPNA

Sucrose

A. cchroccoccum 7B

A. vinelandiiOPN

Glucose

R. eutropha

Sucrose + Molasses Glucose

Sucrose

A. lata

E. coli XL-1

Carbon source Acetic acid

Bacteria strain C. necator

When aeration is low When aeration is high C/N ratio ¼ 10 in a batch culture C/N ratio ¼ 18 in a batch culture

pH of between 6.0 and 7.0

Culture conditions C/N ratio ¼ 4 C/N ratio ¼ 72 C/N ratio ¼ 20, C/P ratio ¼ 1.9, D.O. concentration ¼ 20%, culture residence time in stationary phase ¼ 1 h C/N ratio ¼ 8, C/P ratio ¼ 1.9, D.O. concentration ¼ 20%, culture residence time in the stationary phase ¼ 1 h C/P ratio ¼ 8, C/N ratio ¼ 10, D.O. concentration ¼ 20%, culture residence time in the stationary phase ¼ 1 h C/N ratio ¼ 10, C/P ratio ¼ 1.9, D.O. concentration ¼ 20%, culture residence time in the stationary phase ¼ 1 h Oxygen concentration ¼ 21% Oxygen concentration ¼ 50% Oxygen concentration ¼ 65% Oxygen concentration ¼ 75% Oxygen concentration ¼ 100% 2% (w/v) sucrose only and sucrose supplemented with molasses

Table 1.6 Effect of different culture conditions on the molecular weight and content of PHB accumulated

27 46

2076 894

67 62 85 85

60 32–35

n.m 80.8 99.8 51.2 3.4 74–79

35

596

102.1 134.4 111.35 125.52 71.9 1200– 1600 590 2000– 2500 2020 1010 2300 6600

15

PHB content (%) 50

Mw (kDa) 820 520 2576

Castillo et al. (2017)

Bocanegra et al. (2013) Peña et al. (2014b)

Myshkina et al. (2008)

Penloglou et al. (2012)

Refs Wang and Yu (2001)

24 S. I. Magagula et al.

n.m stands for not mentioned

C/N ratio ¼ 10 in a fed-batch culture with a one pulse feeding strategy C/N ratio ¼ 14 in in a fed-batch culture with a one pulse feeding strategy C/N ratio ¼ 18 in a fed-batch culture with a one pulse feeding strategy C/N ratio ¼ 14 in a fed-batch culture with a two pulse feeding strategy C/N ratio ¼ 18 in a fed-batch culture with a two pulse feeding strategy 60 74 80 71 77

1900 3700 5300 3700 3100

1 The Production and Applications of Microbial-Derived Polyhydroxybutyrates 25

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Regardless of how the PHB is formed or accumulated, the extraction of PHB from the cell cytoplasm involves the rupture of the bacteria cell and the subsequent elimination of the layer of protein wrapped around the PHB granules. However, other PHB extraction methods which involve selectively dissolving PHB granules using a good solvent and subsequently precipitating the dissolved PHB have been alternatively used. This method of extraction is called the solvent extraction method (Kunasundari and Sudesh 2011). Solvent extraction is one of the most commonly used technique for extracting of PHB from cellular biomass. This is a result of the simplicity, rapidity and the ability of the technique to allow for the assessment of PHB accumulation. The solvent extraction method involves a two-step sequential procedure. The first step involves changing the permeable nature of the cell membrane to improve the release and dissolution of the PHB. In the second step, non-solvents are used to precipitate the PHB that has been dissolved in the first step (Jacquel et al. 2008). The dissolution step of the solvent extraction method uses solvents such as chlorinated hydrocarbons like chloroform and 1,2-dichloroethane (Ramsay et al. 1994) as well as cyclic carbonates like 1,2-propylene carbonate and ethylene carbonate (Carpine et al. 2020). Contrastingly, the precipitation step of the solvent extraction method uses non-solvents such as ethanol and methanol (Ramsay et al. 1994). The solvent extraction method has a few advantages as compared to other extraction methods. One of its main advantages amongst others is its extraction efficiency. Another advantage is that the solvent extraction method is capable of eliminating bacterial endotoxin and therefore eliminates the chances of significant polymer degradation (Jacquel et al. 2008). Therefore, this method gives the possibility of obtaining PHB with high purity and significantly high molecular weights. Unfortunately, the application of the solvent extraction method on a larger scale is limited by the use of solvents which are not environmentally friendly (Carpine et al. 2020). Table 1.7 shows a summary of all the solvents that are used during the solvent extraction method. The PHB yield and purity results obtained after the use of the different solvents is also shown. Furthermore, the digestion method has been used as an alternative extraction method to the solvent extraction technique (Carpine et al. 2020). According to the digestion method, the extraction of PHB is achieved by the dissolution of the cellular material wrapped around the PHB. Table 1.8 shows a summary of the digestion methods applied during the extraction of PHB. The purity and the PHB yield are also shown in the table. In order to prevent environmental pollution, a simple, practical, efficient, costeffective and environmentally friendly PHB extraction method that uses non-halogenated solvents is required (Carpine et al. 2020). As a result, several PHB extraction methods have been reported in this regard. For instance, the use of ionic liquids (ILs) to recover PHB accumulated in cyanobacteria by dissolving the cyanobacteria and retaining the PHB has been reported in literature (Kobayashi et al. 2015). Now, ILs are gaining much attention for use as novel green solvents. Their melting point is below 100
C. ILs also have interesting properties such as high thermal stability and high ionic conductivity which are not found in conventional organic solvents (Kobayashi et al. 2015). Other authors have also reported the use of

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Table 1.7 Summary of the solvents used by the PHB solvent extraction method Solvents used Chloroform Chloroform 1,2-propylene carbonate Methyl tert-butyl ether Butyl acetate Non-halogenated acetone/ ethanol/propylene carbonate Acetone

Bacteria strain Bacillus cereus SPV Cupriavidus necator DSM 545 Cupriavidus necator DSM 545 Pseudomonas putida KT2440 C. necator C. necator P. putida GPo1

Results Purity ¼ 92%; Yield ¼ 31% Purity ¼ 95%; Yield ¼ 96% Purity ¼ 84%; Yield ¼ 95% Yield ¼ 80– 85% Purity ¼ 99; Yield: 96 Purity ¼ 93%; Yield ¼ 92% Yield ¼ 94%

Refs Valappil et al. (2007) Fiorese et al. (2009) Fiorese et al. (2009) Wampfler et al. (2010) Aramvash et al. (2015) Fei et al. (2016) Elbahloul and Steinbüchel (2009)

the genetic engineering approach to liberate intracellular PHB granules. Jung et al. (Jung et al. 2005) developed an Escherichia coli strain that was capable of producing sufficiently high yields of PHB by manipulating the inoculum size and medium composition. After a spontaneous disintegration of the bacteria cells, the PHB granules were delivered into the culture medium. The use of solvents that are not chlorinated such as anisole (Rosengart et al. 2015) or solvents that are not halogenated such as butyl acetate (Wang et al. 2015) has also been considered as environmentally friendly PHB extraction method. Furthermore, the use of a solventfree technique such as enzyme digestion has also been considered as an environmentally friendly route for the extraction of PHB. Martino et al. (Martino et al. 2014) used a combination of the enzyme Alcalase and an aqueous medium of sodium dodecyl sulphate (SDS) and ethylenediaminetetraacetic acid (EDTA) for the extraction of PHB granules from the biomass of Cupriavidus necator DSM 428 where used cooking oil (UCO) was used as a source of carbon. The extracted PHB granules had a purity of >90% and no crystallization.

1.7

Applications of PHB

The most outstanding feature of PHBs is that they are 100% biodegradable. However, the biodegradability occurs when the materials are exposed to biologically active environments such as in soils, seawater or freshwater as well as in aerobic and anaerobic compost (Telles et al. 2011). Now, biologically active environments are environments where the PHB materials come into contact with degrading microorganisms (Woolnough et al. 2010). In addition, environments such as in activated sludge (Corrêa et al. 2008) and in sanitary landfills, in which the materials can be discarded without causing any harm to the environment, are also considered as biologically active (Casarin et al. 2013).

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Table 1.8 A summary of the digestion methods for the extraction of PHB Digestion extraction method Surfactant

Materials used SDS Palmitoyl carnitine

Surfactant-sodium hypochlorite

Sodium hypochlorite

Surfactant chelate

Dispersion of chloroform and sodium hypochlorite

Proton-selective dissolution Enzymatic digestion

Mechanical disruption

SDS-sodium hypochlorite Triton X-100-sodium hypochlorite Sodium hypochlorite

Azotobacter chroococcum G-3 C. necator DSM 545

Results Purity ¼ 99%; Yield ¼ 89% Degree of lysis ¼ 56– 78% Purity ¼ 98%; Yield ¼ 87% Purity ¼ 98%

Refs Choi and Lee (1999) Lee et al. (1993) Dong and Sun (2000) Ramsay et al. (1990)

Purity ¼ 86%; Purity ¼ 93%

Sodium hypochlorite

C. necator recombinant Escherichia coli Staphylococcus epidermidis

Triton X-100-EDTA

Sinorhizobium meliloti

Purity ¼ 68%

Betaine-EDTA disodium salt Chloroformsodium hypochlorite Chloroformsodium hypochlorite Sulphuric acid

C. necator DSM 545 B. cereus SPV

Purity  96%; Yield ¼ 90% Purity ¼ 95%; Yield ¼ 30%

C. necator recombinant Escherichia coli C. necator

Purity  98%

Hahn et al. (1995)

Combination of enzyme with SDS-EDTA Alcalase mixed with EDTA and SDS Bead mill

P. putida

Purity  97%; Yield  95% Purity ¼ 93%

Yu and Chen (2006) Kathiraser et al. (2007)

Cupriavidus necator DSM 428

Purity  90%; Yield  90%

Koksharova and Wolk (2002) Tamer et al. (1998) Tamer et al. (1998) Ghatnekar et al. (2002)

High-pressure homogenization SDS-highpressure homogenization Spontaneous liberation

Bacteria strain Recombinant Escherichia coli C. necator, Alcaligenes latus

A. latus A. latus Methylobacterium sp. V49

Purity ¼ 95%; Yield ¼ 98%

E. coli

80% autolysis

Hahn et al. (1995) Marjadi and Dharaiya (2014) Lakshman and Shamala (2006) Chen et al. (2001) Valappil et al. (2007)

Toh et al. (2008)

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Furthermore, biocompatibility is another important feature of PHBs. Now, biocompatibility refers to the suitable interaction between a particular material and its biological environment. In simpler terms, the substance is considered biocompatible if it is not rejected by the body and non-toxic waste is generated during assimilation (Dos Santos et al. 2017). PHB is considered a biocompatible material for two reasons. The first reason is that, it is found in the form of low molar mass PHB in the bloodstream. The second reason is that it produces 3-hydroxybutyric acid upon degradation, which is a common metabolite found in living things (Dos Santos et al. 2017). Due to their biodegradability and biocompatibility, PHBs find many applications in the biomedical, packaging, tissue engineering and drug delivery fields (Dos Santos et al. 2017). Some of the applications of PHBs are shown in Table 1.9. PHBs have other features that limit their applications in various fields. For instance, PHBs have a physical ageing effect which leads to their brittle behavior (Dos Santos et al. 2017; Raza et al. 2019). PHBs are also thermally unstable and are therefore susceptible to thermal degradation during processing. This can be attributed to their narrow processing window, which is characterized by the close proximity between their melting and degradation temperatures. Another major drawback is that PHBs flow during processing (Dos Santos et al. 2017, Raza et al. 2019). Therefore, several approaches aimed at improving their mechanical properties and reduction of the production cost of PHB has been reported in literature. These approaches involve the preparation of PHB blends, PHB copolymers and insertion of additives to develop novel PHB-based composites with optimum properties (Dos Santos et al. 2017, Raza et al. 2019). Some of the PHB-based composites used in PHB applications together with their preparation methods are shown in Table 1.9.

1.8

Conclusion

Polyhydroxybutyrates (PHBs) are a class of biopolymers that are synthesized and accumulated by several bacterial strains as a reserve for energy and carbon. The microorganisms produce the PHB under stressful conditions where there is an excess of carbon and an absence of an essential nutrient such as nitrogen, phosphorus, sulphur or oxygen. Furthermore, PHBs are part of a large group of polyesters called polyhydroxyalkanoates (PHAs) which have microbial-derived, lipid-like and waterinsoluble properties. The world is faced with an environmental pollution problem. Petroleum-based plastics are the main contributors to this environmental pollution problem. Due to their 100% biodegradability, PHBs have been used to replace the non-degradable petroleum-based plastics. Their biodegradability combined with their biocompatibility has earned PHBs a place in many industrial applications such as in the biomedical, packaging, tissue engineering and drug delivery fields. Due their high production cost, PHBs are still produced on a small scale. The hike in the production cost is mainly due to the cost of the carbon source materials and extraction processes. The high cost of the carbon source materials is due to their competition with the agro-food market. In order to reduce the carbon source material

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Table 1.9 A summary of the preparation, applications and properties of PHB-based composed PHB-based composite TCP/PHBV, HA/PHBV PHB/ cellulose PHB/PLA/ CNF

PHB/CNCs

PHB/ cellulose cardboard PHBV/ recycled cellulose fibres PHB/HA

PHB/HA PHB/starch

Preparation of composites Compounding, compression molding Grafting by extrusion mechanism Melt mixing, injection molding and compounding Solution casting

Solvent casting and compression molding Melt mixing

Compression molding

Ball milling and hot pressing Melt compounding

PHB/maize starch

Compression molding

PHB/starch

Hot pressing and melt mixing

PHB/chitin

Solution casting

PHB/α-chitin/ chitosan PHB/chitosan

Solution casting Precipitation method

Properties of prepared composites Reduced degradation Temperature and crystallinity Reduced crystallinity and brittleness

Applications Biomedical

Refs Chen and Wang (2002)

Packaging

Wei et al. (2015)

Decreased elongation at break and increased elastic modulus

Packaging

Kiziltas et al. (2016)

Increased tensile strength and Young’s modulus Increased Young’s modulus

Packaging

Seoane et al. (2016)

Packaging

Seoane et al. (2015)

Increased storage modulus and tensile strength

Packaging

Bhardwaj et al. (2006)

Increased crystallinity and decreased decomposition temperature Improved bending strength of 46.6 MPa Improved thermal stability

Bone tissue engineering

Shishatskaya et al. (2006)

Tissue engineering Packing

Zhuo et al. (2010) Zhang and Thomas (2010) Thiré et al. (2006)

Decreased elongation at break and tensile strength Increased tensile strength (5.9  0.1 MPa) and tear strength (44.1  0.2 kJ/m2) Melting temperature of 166–170
C Melting temperature of 155–170
C Tm of 173.2
C and ΔHm of 91.5 J/g, respectively

Packing

Packing

Lai et al. (2006)

Biomedical

Reddy et al. (2015) Ikejima et al. (1999) Chen et al. (2005)

Biomedical Tissue engineering

(continued)

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Table 1.9 (continued) PHB-based composite PHB/chitosan

Preparation of composites Freeze drying and emulsion blending

PHB/chitosan

Injection molding

PHB/PCL

Solution casting

PHB/PCL

Compression molding and compounding

PHB/silk

Electrospinning technique

PHBV/silk

Electrospinning technique

PHB/PLA

Melt mixing

PHB/PLA

Melt compounding

Properties of prepared composites Elastic modulus (8.7  1.6 to 4.9  0.6 MPa) and swelling capability (141.1  5.9 to 119.3  4.3%) Young’s modulus as (1044–2499 MPa), tensile strength as 11  0.2 to 8.5  0.3 MPa and impact strength as 1595  5 to 159  3 kJ/m2, respectively Increase in elongation at break (300%) and tensile strength (28.23 MPa) Elastic modulus (1643  59 MPa) and tensile strength (21.4  0.3 MPa) Elongation at break as 17.10% and tensile strength as 3.81  0.1 MPa Tensile strength as 1.31  0.20 to 5.82  0.50 MPa, Young’s modulus as 56.5  5.7 to 67.7  5.2 MPa and elongation at break as 1.31  0.20 to 5.82  0.50 MPa 58% decrease in tensile strength, ~50fold increase in elongation at break and Tg as 23 to 19
C Decreased tensile strength and elongation at break

Applications Tissue engineering

Refs Cao et al. (2005)

Drug delivery

Rajan et al. (2012)

Bones regeneration

Saeb et al. (2014)

Bones regeneration

Garcia-Garcia et al. (2016)

Tissue engineering

Karahaliloğlu (2017)

Tissue engineering

Yang et al. (2011)

Food packing

D’Amico et al. (2016)

Food packing

Zhang and Thomas (2011) (continued)

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Table 1.9 (continued) PHB-based composite PHB/flax fibre

PHB/kenaf fibre PHB/PEG

Preparation of composites Compression molding and injection molding Compression molding Salt leaching method

Properties of prepared composites Reduced tensile properties

Increased elongation at break and tensile strength Young’s modulus as 35  1 to 110  2 kPa and bulk density as 1.08 g/cm3

Applications Biocomposites development

Refs Barkoula et al. (2010)

Packaging

Kuciel and Liber-Kneć (2011) Castellanos et al. (2003)

Biomedical

CNF cellulose nanofibres, CNC cellulose nanocrystals, PCL poly(caprolactone), HA hydroxyapatite, PHB poly(hydroxybutyrate), PEG poly(ethylene glycol), TCP tricalcium phosphate, PLA poly (lactic acid)

cost, waste materials, CO2 and sunlight have been used as carbon source materials. However, more work needs to be done to reduce the cost of the carbon source materials. For the extraction of PHB, the cheaper solvent extraction method is used. Unfortunately, the solvent extraction method is not environmentally friendly on a large scale. However, the digestion method is therefore used as an alternative to the solvent extraction method. Therefore, in order to reduce the cost of the extraction process, a process that is environmentally friendly, cheaper, simple, efficient and uses non-halogenated solvents needs to be investigated. Furthermore, the industrial application of PHB is limited by its brittleness and narrow processing window which is caused by the close proximity between their melting and degradation temperatures. This limitation is overcome by blending PHBs with other polymers, fibres or additives to form PHB-based composites with optimum properties.

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Zhang M, Thomas NL (2010) Preparation and properties of polyhydroxybutyrate blended with different types of starch. J Appl Polym Sci 116:688–694 Zhang M, Thomas NL (2011) Blending polylactic acid with polyhydroxybutyrate: the effect on thermal, mechanical, and biodegradation properties. Adv Polym Technol 30:67–79 Zhang D, Dechatiwongse P, Hellgardt K (2015) Modelling light transmission, cyanobacterial growth kinetics and fluid dynamics in a laboratory scale multiphase photo-bioreactor for biological hydrogen production. Algal Res 8:99–107 Zhuo ZQ, Dong LM, Wang C, Zan QF, Tian JM (2010) Preparation and mechanical properties of HA/PHB composites. Adv Mater Res 105-106:104–107

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Fungal Exopolysaccharides: Types, Production and Application Ashim Debnath, Bimal Das, Maimom Soniya Devi, and Ratul Moni Ram

Abstract

Exopolysaccharides (EPSs) may be regarded as extracellular metabolites of living organisms (bacteria, fungi, algae, plants and animals, etc.) associated with adaptation, survival and functionalities. Exopolysaccharides (EPSs) produced from fungi have been documented as high worth molecules for the recent few decades. A wide array of fungal exopolysaccharides is reported to exhibit numerous applications in pharmaceuticals, industries, medicine, foods and other sectors, viz. scleroglucan, botryosphaeran, pestan, pullulan, etc. Although fungal EPSs are exceptionally relevant, to date data concerning fungal biosynthesis is scant and a broad chase for new fungal species which could create novel EPSs is as yet required. As a rule, the molecular weight disparities and sugar syntheses of fungal EPSs are needy to culture medium synthesis and distinctive states of being given during maturation. An accentuation is likewise given to drilling down various parasitic strains that can deliver effective EPSs. The variable synthetic and biochemical engineering that describes an EPS preset its organic usefulness and potential biotechnological benefits. It is agreeable to hereditary, biotechnological

A. Debnath Department of Genetics and Plant Breeding, A. N. D. University of Agriculture and Technology, Ayodhya, Uttar Pradesh, India B. Das Department of Genetics and Plant Breeding, Uttar Banga Krishi Vishwavidyalaya, Dakshin Dinajpur, West Bengal, India M. S. Devi Department of Entomology, Rani Lakshmi Bai Central Agricultural University, Jhansi, Uttar Pradesh, India R. M. Ram (*) Department of Plant Pathology, SGT University, Chandu Budhera, Gurugram, Haryana, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_2

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and biochemical mobility for sought bioactivity or application during their production and extraction. A brief idea regarding fungal EPS is an insight into this chapter. The overall chapter describes the concept of fungal EPS, their production confirmation and wide applications. Keywords

Fungal exopolysaccharide · Scleroglucan · Botryosphaeran

2.1

Introduction

Polysaccharides of high molecular weight have been utilized as additives for emulsifiers (a substance that stabilizes an emulsion, in particular, an additive used to stabilize processed foods), flocculants (clarifying agents removing suspended solids from liquids by inducing flocculation), coatings or films (direct coatings to food into a film used as a food wrap without changing the original ingredients), gelling agents (substances providing texture to foods through gel formation), stabilizers (additives helping in the preservation of foods structure) and thickening agents (substances which increases fluid viscosity without substantially changing its other properties) used in many food products. Polysaccharides obtained from plants cellulose or pectin and seaweeds carrageenan or alginate (Kleerebezem et al. 1999). Interest in polysaccharides from fungi has risen in recent years due to the high productivity and low production costs. Polysaccharides bioactive molecule is produced by many microorganisms such as bacteria, fungi and yeast (Wang et al. 2012). Among the different microbial polysaccharides, one of the most important types is exopolysaccharides which have many advantages over intracellular polysaccharides and its isolation process is easy in a short period from the microorganism (Mahapatra and Banerjee 2013). Exopolysaccharides (EPS) are mainly extracellular polymers of carbohydrate which are synthesis by microorganisms and utilize them in cell outside. They do not serve as reserve materials, making them different from various other intracellular polymeric inclusions. Exopolysaccharides are fitted to the cell surfaces and play a major role in protecting against phagocytosis and phage attack. They also act as barriers to diffusion and bind the metal ions (Cu, Pb, Zn, Co, etc.) which help microorganism under adverse conditions. Microbial origin EPSs, including fungal EPSs, are a justifiable alternative to marketed polysaccharides. The microbial-based polysaccharides are almost identical to those currently used gums (Sutherland 1996a, b). These polysaccharides have achieved importance from the last few decades based on their different applications in various fields. The term ‘exopolysaccharide’ was first described as high molecular weight polymers of carbohydrate produced by marine bacteria (Sutherland 1982). Exopolysaccharides composed of repeated sugar moieties may be associated with other molecules like proteins, organic, inorganic compounds, lipids, DNA and metal ions. The definite actions of EPS and exact function depend on ecological niches and structural units of the concerned microorganisms (Mishra and Jha 2013). Substantial

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progress is being carried forward to discover and develop new microbial EPSs with novel and interesting industrial applications (Nicolaus et al. 2010). EPS are used as emulsifiers, binders, stabilizers, coagulants, gelling agents, lubricants, thickening and film-forming agents in food and pharmaceutical industries. Microbial exopolysaccharides served many functions in the microbial cells and are differentiated into three main types mainly structural, intracellular and extra or exopolysaccharides. Chitin was the first polysaccharide of carbohydrate polymer discovered in edible mushrooms and the production of extracellular polysaccharide by microorganisms first reported in 1861. However, microorganisms are known for their ability to synthesize exopolysaccharides (EPS), both homo- and heteropolymers with different structural complexities (Seviour et al. 1992; Sutherland 1994). These microbial exopolysaccharides represent an attractive alternative to synthetic polymers and represent biodegradable polymers (Dave et al. 2016) which is ideal to replace petro-based polymers (Meybod and Mohammadifar 2015; Castillo et al. 2015; Moscovici 2015; Schmid et al. 2016). Similarly, fungal exopolysaccharides far better alternative to exopolysaccharides produced by plant or macroalgae (Freitas et al. 2011; Moscovici 2015; Meybod and Mohammadifar 2015). Several metabolites have been reported to be produced from fungi. The important among these metabolites are enzymes, exopolysaccharides, antibiotics, etc. Interest in these natural metabolites is significant because of their medical, industrial and agricultural importance. Different fungal EPS with interesting biological activities, from Agaricus blazi, Ganoderma lucidum, Cordyceps sp., Grifola frondosa and Lentinus edodes through submerged cultures (Yang and He 2008). A glucan was extracted from aqueous extract of fruiting bodies of Pleurotus florida (Rout et al. 2005). EPS of medicinal importance has been extracted from Pleurotus (Fr.) P. Karst and Lentinus edodes (Berk.), Phellinus rimosus, Ganoderma lucidum, Pleurotus pulmonaris and Pleurotus florida and are found to possess profound antioxidant and antitumor activities (Elisashvili et al. 2009; Ajith and Janardhanan 2007). Biologically active extracellular exopolysaccharides and intracellular polysaccharides in fruit bodies, cultured broth and cultured mycelium (Wasser 2010) of many fungi are useful to pharmacological industries in the aspects of the antioxidant property, hypoglycemia, anti-inflammatory, antitumour, immuno-stimulating and ROS activity (Chen et al. 2012; Hu et al. 2006a, b; Wu et al. 2010. Despite having immense potential for EPS production, only medicinal aspects of these fungi have been explored. They are capable of producing EPS of high commercial value and can be used for various industrial applications.

2.2

Sources of Fungal Exopolysaccharides

Fungal EPSs have been identified as highly useful bio-macromolecules. EPSs like scleroglucan, botryosphaeran and pullulan have many uses in food, medicine, pharmaceuticals industries, etc. The fungal EPS are extremely pertinent and identification of novel fungal species for the biosynthesis of potential EPSs is still required.

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Table 2.1 Source of fungal exopolysaccharide Sl. no. 1 2 3 4 5 6 7

Fungus species Filamentous fungi, Candida albicans, Zygosaccharomyces rouxii Sclerotium rolfsii Schizophyllum commune Botryosphaeria rhodina Cryptococcus neoformans, Aspergillus fumigatus Aureobasidium pullulans, Pullularia pullulans Pleurotus ostreatus

Exopolysaccharide Chitin and chitosan Scleroglucan Schizophyllan Botryosphaeran Galactosaminoglucan Pullulan Pleuran

Variations in molecular mass and composition of sugar in fungal EPSs depends on the composition of culture media and various physical conditions during fermentation. EPS production through submerged fermentation by different fungal strains, including Agaricus blazi, Ganoderma lucidum, Cordyceps sp., Grifola frondosa and Lentinus edodes had been reported, with unique biological activities. Presently, a large number of fungi including higher basidiomycetes, lower filamentous fungi and yeasts from various ecological habitats are found to produce EPSs in laboratory conditions. However, many remain unexplored or under-investigated. Some of the important fungal exopolysaccharides sources (Table 2.1) and their structure are presented in the following:

2.2.1

Chitin/Chitosan

The cellulose analogue chitin constitutes one of the most abundant glycans. It occurs in different crystalline polymorphic forms. Among them α-chitin is on the most common forms of chitin. Chitin is a long-chain polymer of a derivative of glucose, N-acetylglucosamine. The primary source of chitin is cell walls of fungi and exoskeletons of insects and different arthropods like lobsters, shrimps, crabs, etc. Chitins are chemically modified to be used in food processing as edible films and as thickeners and stabilizers chitin and chitosan have been used in paper strengthening (Hosokawa et al. 1990). Chitosan is fully or partially N-deacetylated derivatives of cellulose. Chitosan is made up of β (1-4)-linked 2-acetamido-2-deoxy-β-D-glucose (N-acetylglucosamine) (Fig. 2.1). Chitosan has received considerable attention recently (Sanford and Hutchings 1987). Chitosans are the major elements derived from arthropods shells including that of shrimps, crabs, lobsters and insects. They are also formed extracellularly by the cell walls of fungi and brown algae. Chitosan-derived biomaterials have received considerable attention as an antimicrobial, functional, renewable, non-toxic, biocompatible, bioabsorbable and biodegradable biopolymer agent (Biagini et al. 1991; Zhang et al. 2010).

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Fig. 2.1 Structure of (a) chitin showing N-acetylglucosamine linked through β-(1!4)-linkage and (b) chitosan comprising of β (1-4)-linked D-glucosamine as deacetylated unit and N-acetyl-Dglucosamine as acetylated unit

Fig. 2.2 Structure of scleroglucan made by a β-(1, 3) backbone linked with D-glucosyl groups linked by β-(1, 6) linkages

2.2.2

Scleroglucan

It is a natural polysaccharide produced by the fungus Sclerotium rolfsii. Structurally is a β-(1, 3) linked with D-glucosyl groups linked via β-(1,6) linkages (Fig. 2.2) and is stable at low pH and high temperature. Scleroglucan is used for the improvement of drug delivery system (Coviello et al. 2005). It is acceptable in making of sustainedrelease tablets and ocular formulations. It shows antiviral property against Herpes Virus and is used in formulations for agriculture. This polysaccharide is mainly used as an industrial viscosifier for the enhanced oil recovery. It has been reported for its antiviral and antitumor activity.

2.2.3

Schizophyllan

The main producer of this polysaccharide is Schizophyllan commune. The average molecular weight is about 500,000 Da (Itou et al. 1986). Schizophyllan is a β-1,

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Fig. 2.3 Structure of schizophyllan showing a β-1, 3 glucan with β-1, 6 branching

Fig. 2.4 Structure of botryosphaeran comprises of β (1, 3) backbone and β (1, 6) branched residues of glucose

3 glucan with β-1, 6 branching structure (Fig. 2.3). This polysaccharide is known for its capability for stimulating the immune system, hold metals in water, help in delivering drugs and making of nanofibers.

2.2.4

Botryosphaeran

Botryosphaeran is an exopolysaccharide which is produced by Botryosphaeria rhodina a ligninolytic and ascomycetous fungus that makes the culture mediumhigh in viscosity when grown on glucose as the main source of carbon. It is capable of forming a strong gel. It is a potent immunomodulatory and shows an antimutagenic effect. The triple-helical conformation of the polysaccharide is stabilized by hydrogen bonding between hydroxyl groups of the polysaccharide chain, which is the main reason for the stable behaviour of these macromolecules (Fig. 2.4).

2.2.5

Glucuronoxylomannan

Glucuronoxylomannan is a major capsular fungal polysaccharide produced by Cryptococcus neoformans. Glucuronoxylomannan is a high molecular weight polysaccharide that is shed during cryptococcosis and very important for immune

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Fig. 2.5 Structure of glucuronoxylomannan consisting of two repeating polysaccharide units

Fig. 2.6 Structure of pullulan consisting of maltotriose units with alternating α-(1, 4) and α-(1,6) linkages

response. It also inhibits the entrance of HIV in TZM-bl cell lines. The interaction of glucuronoxylomannan with monocytes or macrophages regulates the killing process, resulting in the reduction of superoxide anion (Monari et al. 2003) and decreased proinflammatory cytokines (Vecchiarelli et al. 1995); alongwith supression of IL-12 (Retini et al. 2001) (Fig. 2.5).

2.2.6

Pullulan

Pullulan is a linear water-soluble polymer isolated from Pullularia pullulans and Aureobasidium pullulans. The molecular weight of pullulan varies from 10–400  103 Da and highly soluble than amylose. Pullulan is a soluble homopolysaccharide comprises of maltotriose units with alternating α-(1, 4) and α-(1, 6) linkages (Fig. 2.6). Generally, pullulan is used in the agri-food industry as viscosity stabilizers, thickeners and biodegradable preparation, edible plastic materials and non-toxic. Since pullulan is only partially degraded by the human amylases, it is utilized as dietary fibre or a prebiotic substance.

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Production Process of Polysaccharides from Fungi

Among all the parameters affecting polysaccharides production, the techniques or the production process has a huge impact on producing polysaccharides qualitatively and quantitatively. The outer cell surfaces of fungi are the main source of polysaccharides. For production of fungal exopolysaccharides different fermentation method are practised under controlled condition. Generally, two important processors are deployed for maximum fungal biomass production and for the quality end product.

2.3.1

Solid-State Fermentation (SSF)

This technique provides good results mostly for the biotechnological fungal products, for example, enzymes or other secondary metabolites (Saqib et al. 2010). One of the important factors for choosing this method is that they are relatively cheaper when the raw material is used as the main subtract (Castilho et al. 2000). Singhania et al. (2009) suggested that this substrate provides the best growing condition for the fungi to grow for producing the polysaccharides. However, he suggested that the limitation of this process lies with the biomass separation for most fungi. Nie et al. (2005) proposed a method for polysaccharides quantification using high-performance liquid chromatography with refractive index detection (HPLC-RI) for comparison of glycoproteins collected from tea species from various locations. Solid-state fermentation overall is the best method of producing fungal biomass as it is a cost-effective process and can yield maximum quantities of fungal biomass. Also for the production of foods that contain polysaccharides derived from fungi such as glucomannans produced from A. subrufescens, this procedure can be utilized to consume as whole biomass obtained through the extension of functional foods.

2.3.2

Submersed Fermentation

This method generally provides more productivity of polysaccharides than the solidstate fermentation as the procedure is subjected to those fungi which have the potentialities to produce a large volume of biomass compared to others and thereby producing a large number of polysaccharides. This method is mostly used for the pharmaceutical area as they require more precise fungal growth condition such as physical, chemical, nutritional factor, a uniform yield of biomass and quality polysaccharides yield (Fazenda et al. 2008). Generally, two types of bioreactors are used for cultivation of fungal biomass in submersed fermentation method, viz. stirred-tank bioreactor which is applied under the aerobic condition and is mostly used in the industrial sector. The other one is airlift and bubble column bioreactor which is operated without mechanical agitators. The disadvantage that lies with the stirred-tank is the stirring requirement to reach ideal biomass of fungi sometimes causes damage to the cells. Apart from that they

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are more expensive and have much lesser resistant than later. The work on the airlift bioreactor is done by Camelini et al. (2013) on A. subrufescens and bubble column by Kawagoe et al. (2004) on the same species was 8 times lower average productivity (0.095 g/L-day) on the latter than the former which has 0.76 g/L-day subject to placement at same airflow rates. The production of polysaccharides mostly depends on right species selection, cultivation method, extraction and purification for final successful results of interest. With the gaining of popularity in today’s world, this technology will help future research with the production of quality product needed by different sectors.

2.4

Compositions of EPS Produced by Different Fungi

The fungal EPSs composition differs from pure sugars to sugars combined with a second unit. This second unit can be a protein, phosphate, sulphate or amine. In homopolysaccharides, glucose is the only monomer but in case of Ascomycota and Basidiomycota EPSs, they are mainly heteropolysaccharides where different types of sugar units were found in fungal EPSs such as glucose, galactose, mannose, fucose, xylose and rhamnose. Different fungi synthesize same monosaccharide units which are the composition of EPSs (Table 2.2). The chemical properties and structures of EPS such as the molecular weight, monosaccharide composition, linkage type and the conformation chain were evaluated with the help of different chromatography technology such as GLC, TLC, HPLC and also with various spectrum analysis like FTIR, 1D and 2D NMR spectroscopy and GLC-MS (Kozarski et al. 2012; Osinska-Jaroszuk et al. 2014; Silveira et al. 2015).

2.5

Parameters Affecting Polysaccharides Production

Polysaccharides production and usage in today’s world have gained a tremendous market due to its wide range of beneficial applications. They form an important constituent in several industrial purposes in the form of feedstock, different types of chemicals material production and also energy. Apart from these various sources of polysaccharides viz., plant, animals and microorganisms, the former contributes most for increasing demand of the market. It has been estimated to produce 90% of polysaccharides only through the vegetable biomass. Among the microorganism producing polysaccharides, fungi are the dominant producers and are the most studied area for the production of polysaccharides. Polysaccharides production from fungal biomass has gained a lot of interest among the researcher as they are most predominant on the cell wall and shares equal relevant physico-chemical and structural properties that have wide range of applications. However, the production process of polysaccharides from fungal biomass varies with that of the other sources and several factors affect the production process.

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Table 2.2 Compositions of some fungal exopolysaccharide Organism Aspergillus niger

EPS composition Glucose

Nigrospora oryzae var. glucanicum Aspergillus alliaceus

Glucose Glucose, Galactosamine, Galactose, Acetate

Drechslera spicifera

Glucose

Aspergillus parasiticus Aspergillus sp. Y16

Glucose, Galactose, Acetate, Galactosamine, Phosphate Galactose, Mannose

Candida boidinii

Glucose, Mannose

Cordyceps sphecocephala Cryphonectria parasitica Cryptococcus laurentii AL100 Cryptococcus neoformans

Glucose, Galactose, Mannose, Protein

Stemphylium sp.

1st type—Glucose, Galactose, Mannose; 2nd type—Glucose Glucose, Galactose, Arabinose, Mannose, Rhamnose 1st—Glucuronic acid, Xylose, Mannose; 2nd— Galactose, Xylose, Mannose; 3rd—Mannose, Protein Glucose, Mannose

Antrodia camphorata Elsinoe leucospila

–

Epicoccum nigrum Ehrenb. ex Schlecht Fusarium solani SD5 Isaria farinosa BO5

Glucose

Lachnum sp. YM261 Cyttaria harioti Phomopsis foeniculi

Glucose

Galactose, Rhamnose Glucose, Mannose, Galactose, Uronic acid, Protein (4.40%) Glucose Glucose

Penicillium varians

1st type—Rhamnose, Mannose, Galactose; 2nd type—Mannose Glucose, Galactose

Pestalotia sp. 815

Glucose

Phanerochaete chrysosporium

Glucose

References Sutherland (1996a, b) Sudhakaran and Shewale (1988) Miranda and Leal (1981) Aouadi et al. (1992) Ruperez and Lal (1981) Chen et al. (2011) Petersen et al. (1990) Oh et al. (2007) Forabosco et al. (2006) Pavlova et al. (2011)

Mahapatra and Banerjee (2012) Shu and Lung (2004) Sutherland (1996a, b) Schmid et al. (2001) Mahapatra and Banerjee (2012) Jiang et al. (2008) Sutherland (1996a, b) Corsaro et al. (1998) Jansson and Lindberg (1980) Misaki et al. (1984) Buchala and Leisola (1987) (continued)

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Table 2.2 (continued) Organism Trichosporon asahii

EPS composition Mannose, Xylose, Glucuronic acid

Pleurotus tuberregium Sporobolomyces sp.

Glucose, Mannose

Pleurotus sp.

Glucose, Galactose, Mannose, Protein

Tremella fuciformis Acremonium diospyri Crandall Botryosphaeria rhodina RCYU30101

Mannose, Xylose, Fucose, Protein Glucose

2.5.1

Galactose

Glucose

References Fonseca et al. (2009) Zhang and Cheung (2011) Pavlova et al. (2004) Gutiérrez et al. (1996) Cho et al. (2006) Seviour and Hensgen (1983) Weng et al. (2011)

Nutrient Source

One of the major factor affecting fungal growth and development is the wide range of nutrient sources. The effect of a nutrient on mycelium can lead to overgrowth or even restrict the growth with negative results. It has been found by various researchers that there has been a direct co-relation of polysaccharides production from fungal biomass and the nutrients supplied. Among the nutrient sources studied, most focus has been given to the effect of nitrogen and carbon sources (Wu et al. 2012a, b; Mahapatra and Banerjee 2013).

2.5.1.1 Nitrogen Source The nitrogen source in the growing media has a great influence on fungal growth, development and metabolite productions. Both the organic and inorganic source of nitrogen can be effectively used. However, organic sources have been seen to produce relatively higher polysaccharides than later. NH4H2PO4 when used as nitrogen sources produce a much higher yield of polysaccharides than KNO3 from Tricholoma margolicum. Some of the well-known inorganic sources are ammonium chloride, ammonium sulphate, potassium nitrate, sodium nitrate, diammonium oxalate monohydrate and urea (Mahapatra and Banerjee 2013). Of these ammonium salts are found to produce effective results in polysaccharide production. Few reports have suggested sodium and urea as a better source of nitrogen when Epicoccum nigrum and S.rolfsii fungi are used for polysaccharides production (Farina et al. 1998; Schmid et al. 2001). Even though a very less amount of nitrogen are required by the fungi (1–10 g/L) to produce polysaccharides, on the one hand, their unavailability, on the other hand, may result in a subsequent reduction in fungal growth and thereby less production on the polysaccharides.

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2.5.1.2 Carbon Source In contrast to nitrogen which is needed in a very minute amount, carbon is very much important in polysaccharides production from fungi as they are the important sources that have a direct effect on growth and development of high fungi. The polysaccharides production intensity is much dependent on the types of carbohydrates used, i.e. on carbon sources. Many sources of carbon have been studied by the researchers: glucose, sucrose, fructose, lactose, maltose, galactose, cellobiose, xylose, sorbitol, mannitol, xylitol, etc. Among which glucose, maltose and sucrose have been mostly studied and have given the most influential results in the production of the polysaccharide from fungal biomass. However, apart from these sources, it has been found that different fungi respond to other carbon sources for their growth and development with the production of the desired end product. Different fungi which have been studied for different carbon sources are presented in the following Table 2.3. Besides the sources of carbon, the concentrations of carbon also play a vital role in the growth and development of fungal biomass and ultimately on polysaccharide production. On average, it has been found that 30–60 g/L carbon was best to produce polysaccharide from fungi. Few reports have also suggested that combined carbon source can enhance the polysaccharide where 2 g/L glucose combined with 30 g/L starch increased production of polysaccharide when produced from Sorangium cellulosum.

2.5.2

pH

The pH is one of the important factors affecting the fungal biomass growth that is required for effective polysaccharide production. Their growth varies with the change of pH, with slight variation in it can lead to overgrowth or undergrowth of fungal biomass within a given environment condition. In general, fungi mostly prefer pH ranging between 3.0 and 6.5 (Pavlova et al. 2011, Luo et al. 2009, Wu Table 2.3 List of fungi studied for different carbon sources Fungi 1. G. lucidum 2. Inonotus levis 3. Phellinus robustus 4. Cerrena maxima 5. Phellinus igniarius 6. Trametes versicolor 7. Agaricus nevei 8. Paecilomyces japonica 9. L. edodes 10. Pleurotus sp. 11. Sclerotium rolfsii (ATCC2011126)

Carbon source Glucose Glucose Glucose Maltose Maltose Maltose Mannitol Mannitol Sodium gluconate Sodium gluconate Sucrose

References Elisashvili et al. (2009)

Bae et al. (2001) Elisashvili et al. (2009) Farina et al. (1998)

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et al. 2012a, b). Besides these neutral pH loving fungi, several other fungi prefer mostly neutral or alkaline pH for polysaccharide production (Kim and Yun 2005; Pokhrel and Ohga 2007). Once the study has suggested a direct relation of polysaccharide production to initial pH where the mycelia growth at an earlier stage was seen faster, however on the later stage there was no clustering of mycelia which resulted in a decrease in mycelia biomass formation and final accumulation of polysaccharides. An example to the effect of pH on the production of polysaccharide can be seen on the studies of Shu and Lung (2004) where they found the variation in polysaccharide production with different molecular weight (Mw) by Antrodia camphorata with variation in pH. They found that at lower pH medium-high Mw polysaccharide was produced in low amount and at high pH, medium-low Mw polysaccharide was produced with high yield. Another study on Mucorrouxii by Abdel-Aziz et al. (2012) showed that the polysaccharide production gradually increased with increase in pH. A work on A. subrufescens showed that the yield of mycelium was maximum when placed in the pH initially at 6.06 and incubated at a temperature of 27  C (Lin and Yang 2006). A slight change in pH with increased polysaccharide production was also reported by Fan et al. (2007) where pH 6.1 and temperature 30  C was suitable for mycelial yield. However, some of the reports also suggested that the pH of about 5.6 and 6.9 and a temperature of about 24.6  C and 20  C was best for maximum polysaccharides production (Hamedi et al. 2007).

2.5.3

Temperature

Temperature is relatively an important factor required for fungal growth and development which in turn affects the polysaccharide production. Different fungi respond to varying temperature for the production of polysaccharides where maximum polysaccharide production from fungal strain lies within the range of 22  C to 30  C (Mahapatra and Banerjee 2013). Apart from these few fungal strain have been reported to respond well at 20  C for polysaccharide production. Xiotong et al. (2012) found that at 25  C T. mongolium responded well and yield maximum polysaccharide. However, with slight variation in above temperature, the mycelia did not respond well to grow resulting in low yield of polysaccharides production.

2.5.4

Size and Age of Fungal Inoculum

The different physiological properties of fungus also play a significant effect on the production of the polysaccharide. Among them, the size of the plant and the age of the fungus in the culture have a tremendous effect on the fungal growth and development (Glazebrook et al. 1992). In other words, if the size of the inoculum is taken smaller it may lengthen the fermentation period, which on the other larger size inoculum may overcome disadvantage because the large inoculum may consume the nutrient quickly which helps in the growth of fungal biomass and thereby producing the polysaccharides preventing the bacterial infection during the

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fermentation process. Xiaotong in his experiment concluded that out of different inoculum, viz., 5, 8, 10, 12 and 15% v/v, only 10–12% inoculum size was seen to produce better polysaccharides production. Age, on the other hand, have a direct effect on the fungal mycelial growth, as over-aged fungal colony may take more time to reach the log phage as compared to the fresh culture provided the same nutrient media and temperature.

2.5.5

Fungal Material Preservation

Storage of fungal material plays an important role in the production of quality polysaccharides. As the condition of the fungal material before and after the preservation directly influence the growth of the fungi. Different type of techniques has been used by various researchers previously but for fungal storage, one can store them depending on the duration of time as follows:

2.5.5.1 Short-Term Preservation It includes maintenance and preservation of culture for a period of short time (less than 1 year). This method is simple, cost-effective and labour intensive. Besides these, it has several disadvantages of frequent checking of contamination, morphological or physiological change of fungi with time. For fungal short term storage, the use of mineral oil as well as distilled water is most popular and widely used for the preservation (Richter 2008; Croan et al. 1999). 2.5.5.2 Long-Term Preservation This technique is mainly used to preserve the fungal culture for long period (1–3 years or even more duration). This method generally overcomes the disadvantages of short term storage. The main logic behind this technique is to arrest growth and metabolic activities for long-term preservation. For fungal longterm storage lyophilization and cryopreservation in liquid nitrogen are mostly used (Croan et al. 1999; Colauto et al. 2012a, b). The choice for opting the method of preservation depends upon several factors. After preservation, the fungal culture must be contamination-free apart from producing a pure mycelium as preserved it must be able to grow and multiply producing successful biomass for polysaccharide production. Also, the factors such as biological activity in the end product are one of the major concern before opting for any method of preservation as some preservative chemicals or substracts may interact with the chemical structure of the polysaccharides present in the cell wall of mycelia which may alter or change its main structural chain also its lateral end chain or the arrangement of its branching degree, which may have an impact in quality polysaccharide production (Lavi et al. 2010; Mantovani et al. 2008). Few examples in case of Agaricus subrufescens. While preserved in cryopreserving with DMSO, it was found to respond differently, only 40% was recovered when frozen and maintained at 196  C for 1.5 years (Colauto et al. 2012a, b). Attenuation of fungal activity is one of the important factors as some of the fungi have the capabilities to

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produce a large number of secondary metabolites as agaritine when their growth are arrested during preservation placed in the mycelia for growth may affect the fungal species through their mutagenic effect (Walton et al. 1997). Maheshwari and Navaraj (2008) found that the storage of most fungi can be done at a temperature ranging between 4  C and 12  C for 6–8 months; however, the difficulties pertaining this can be the induction of senescence which may lead to the failure of progressive potential mycelial growth, induction of the mutation, hindrance of growth which may lead to fungal death. Some researchers also suggest that the use of activated charcoal for preserving A. subrufescens is a quick, easy and cost-effective method as it was found that it did not affect much on morphological or genetical changes even when stored for 1 year.

2.5.6

Additives

There have been several reports that the addition of some additives resulted in maximum EPS production. Such finding has been presented with the addition of fatty acid, vitamins, vegetable oils and surfactants (Yang and He 2008; Xiao et al. 2010; Zhang and Cheung 2011; Yang et al. 2000). In addition to these use of glutamic acid, biotin, thiamine (Lee et al. 2001), soybean oil (0:1) in culture media, palmitic acid and oleic acid showed increased production of EPS when Shiraria bambusicola was used. Apart from these some of the surfactant which has widely been tested such as Tween 80 and Span 80 induced maximum EPS production when 3 g/L is added. However, Hsieh et al. (2008) found that the same surfactants even though induced the fungal growth but EPS production was reduced significantly in G. frondora. He also found that at a stationary phase when olive oil (0.5%) is added the fungus was able to produce maximum EPS. Furthermore, addition vitamins like Vit A & D in media, 0.1% (v/v) thiamin & nicotinic acid at the rate of 0.1% (v/v) helped to increase exopolysaccharides production by Oudemansiella radicata and Antrodiac cinnamomea, respectively (Lin and Chen 2007; Xiao et al. 2010).

2.6

Different Applications of Fungal Polysaccharides

Fungal EPSs being one of the prime components of microbial EPSs have gained wide significance in the course of a recent couple of decades, as few studies demonstrated their applicability in various fields. Besides this, production of EPSs is simpler and a large number of these products can be developed in a shorter period in comparison with products of plant or algae. Fungal EPSs have got few applications in the food, cosmetics and drug enterprises beside others. Fungal EPSs, like pullulan, scleroglucan and botryosphaeran, has many important applications in fields of food and pharmaceutical industries. Among the various applications, EPSs are used as biosurfactants, bioemulsifiers and bioflocculants due to their structural and functional diversity. EPS-based bioflocculants are mostly

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Fig. 2.7 Multiple applications of fungal EPs in different fields

composed of polysaccharides, proteins, glycoproteins, nucleic acids and lipids. The process where stable aggregates are formed by extracellular polymers released by living cells is termed as bioflocculation. Exopolysaccharides as bioflocculants have gained increased attention as they possess ionizable functional groups like amine, carboxyl, acetate, sulphate and hydroxyl groups which make these biopolymers to be effective in the removal of suspended solids, heavy metals and in reducing the turbidity of different types of industrial wastewater effluents. Their use has been well documented in textiles, detergents, raw water treatment, adhesives, petroleum refinery effluent, oil recovery, paper industry and settling of sludge in aerobic and anaerobic treatment systems. An overview of various applications of fungal EPSs is presented in Fig. 2.7. Among the diverse fungal EPSs scleroglucan, botryosphaeran and pullulan are notable for exhibiting multiple roles in various fields. However, a few fungal EPSs have been described for understanding the extent of vivid applications of macromolecules. Pullulan, an EPS of A. pullulans, can be utilized as a thickener, a viscosity stabilizer in the food business, and for designing of non-toxic, biodegradable, palatable plastic materials (Paul et al. 1986). Fungal EPS scleroglucan is utilized mostly for the Enhanced Oil Recovery (Holzwarth 1985). Scleroglucan often is known as schizophyllan under the trade name BIOVIS with bacterial polysaccharides, xanthan gums are usually used in making of drilling liquids with low mud impurities (Hamed and Belhadri 2009). Farwick et al. (2009) reported that scleroglucan possess the characteristic feature of high-water adhering ability on epithelial cells. Other modern utilizations of this EPS

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were in the manufacture of glues, water hues, printing inks and formation of animal feed (Halleck 1969). It is also being applied in the manufacture of cosmetics and different skin health management items, lotion and creams (Halleck 1970, 1972). EPS of Byssochlamys demonstrated the property of Kaolin flocculating conduct. Gomoiu and Catley proposed that the EPS can be utilized for deposition of filaments in downstream expulsion of paper strands from water effluent in paper enterprises and subsequently has reduced efficacy in the paper industry and rivers wherever mechanical effluents were disposed of (Catley 1971). Five yeast strain EPSs and from one yeast-like organism demonstrated drag-lessening action (Petersen et al. 1990). Exopolymer obtained from G. lucidum can enhance the swimming durability of mice by around 10 min and decreased the glycogen content in muscle and liver depletion by 18.5% and 67.2%, respectively (Yang et al. 2001). Fungal-glucans (both intracellular and extracellular) are compelling in advancing health, safeguard from mutations and therapy of ailments like disease, microbial contaminations, diabetes and hypercholesterolaemia (Chen and Seviour 2007; Mantovani et al. 2008). Pestan, a contagious EPS obtained from Pestalotiopsis sp. KCTC 8637, has been used in the treatment of wastewater as a biosorbent of zinc and lead. Every gram of pestan can absorb 60 mg zinc and 120 mg lead (Moon et al. 2006). Similarly, EPS of Aspergillus fumigatus likewise demonstrated sorption proficiency of two substantial metal particles, i.e. lead and copper (Yin et al. 2011). Botryosphaeran produced from Botryosphaeria rhodina appeared to diminish altogether the clastogenic impact of cyclophosphamide-instigated micronucleus arrangement in polychromatic erythrocytes of bone marrow and reticulocytes infringe blood in mice (Miranda et al. 2008). It is also known to be a strong immunomodulator (Weng et al. 2011). EPSs delivered by Akanthomyces pistillariiformis BCC2694, P. tenuipes BCC2656, Cordyceps dipterigena BCC2073 and Phytocordyceps sp. BCC2744 demonstrated to be biocompatible and have the probability to act as a wound dressing material (Madla et al. 2005). EPS of Phellinus baumii Pilat demonstrated direct safe invigorating action on splenocyte proliferative reaction and corrosive phosphatase movement in peritoneal macrophages of mice (Luo et al. 2009). Wang et al. (2011) described the immunomodulatory exercises of EPS delivered by C. sinensis Cs-HK1. Water solvent EPS of Isaria farinosa BO5 demonstrated antitumor and cell reinforcement movement in Kunming mice (Weng et al. 2011). EPS of Fomes fomentarius and Hypsizygus marmoreus demonstrated antitumor action inspected on human gastric disease cells SGC-7901 (Chen et al. 2008; Zhang et al. 2012). EPS of C. sinensis Cs-HK1 has moderate cancer prevention potential.57 EPSs of Gomphidius rutilus, Aspergillus sp. Y16 and Ganoderma resinaceum appeared in vitro opponent of oxidant movement (Kim et al. 2006; Chen et al. 2011; Gao et al. 2012). Fusarium oxysporum DzF17 has been accounted to be an EPS-producing fungus. The EPS indicated elicitor exercises on the development and diosgenin formation in cell suspension culture of Dioscorea zingiberensis (Li et al. 2011). F. solaniSD5 was accounted for to create an extracellular rhamnogalactan that indicated calming and hostile to hypersensitive action in vitro. (Mahapatra and Banerjee 2012). G. frondosa HB0071 EPS demonstrated

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inhibitory impact on grid metallo proteinase-1 articulation in UV-lighted human dermal fibroblasts, and this manner may add to inhibitory activity in photograph maturing skin by diminishing framework metallo proteinase-1-related network debasement framework (Bae et al. 2005). EPS produced by Stropharia rugosoannulata and P. baumii show hypoglycemic movement in streptozotocin actuated high sugar rodents (Zhai et al. 2012). Primer assessments with EPS formed by R. glutinins are hostile to oxidant, antitumor and antiviral exercises (Ibrahim et al. 2012).

2.7

Conclusion

Hurtley et al. (2001) stated that the science and science of carbohydrate research resembles a ‘Cinderella field’, however till date, it doesn’t receive much appreciation as that of genomics and proteomics. This acknowledgement is touched with authenticity, even though microorganisms have been widely employed since the 1940s for the development of assorted biomaterials. On account of EPS creation, notwithstanding, the endeavours were not acceptable to extinguish the hunger for information in this field as of not long ago. Significant increments in the examination of fungal EPS production or their physio-synthetic portrayals by specialists were seen uniquely in the course of the most recent 20 years. By the by, the discoveries are amazing from both the logical edge furthermore, pertinence. There is no uncertainty that EPS and its derivatives have promising possibilities that could be investigated for assorted additions, including public and unfamiliar trade benefits through connecting with worldwide biopolymer market. The expectation is that this pattern will proceed to increment and will enhance both logical information base and will open new avenues in different sectors for encouraging our livelihood.

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Singhania RR, Patel AK, Soccol CR, Pandey A (2009) Recent advances in solid-state fermentation. Biochem Eng J 44(1):13–18 Sudhakaran VK, Shewale JG (1988) Exopolysaccharide production by Nigrospora oryzae var. glucanicum. Enzyme Microb Technol 10(9):547–551 Sutherland IW (1982) Biosynthesis of microbial exopolysaccharides. Adv Microb Phys 23:79–150 Sutherland IW (1994) Structure-function relationships in microbial exopolysaccharides. Biotech Adv 12(2):393–448 Sutherland IW (1996a) Extracellular polysaccharides. In: Rhem HJ, Reed G (eds) Biotechnology, vol 6. Wiley-VCH, Weinheim, pp 615–657 Sutherland IW (1996b) Extracellular polysaccharides. In: Rehm HJ, Reed G (eds) Biotechnology: products of primary metabolism, vol 6, 2nd edn. Wiley-VCH Verlag GmbH, Weinheim, pp 613–657 Vecchiarelli A, Retini C, Pietrella D, Monari C, Tascini C, Beccari T, Kozel TR (1995) Downregulation by cryptococcal polysaccharide of tumour necrosis factor-alpha and interleukin1 beta secretion from human monocytes. Infect Immun 63:2919–2923 Walton K, Coombs MM, Walker R, Ioannides C (1997) Bioactivation of mushroom hydrazines to mutagenic products by mammalian and fungal enzymes. Mutat Res 381:131–139 Wang ZM, Peng X, Lee KLD, Tang JC, Cheung PCK, Wu JY (2011) Structural characterization and immunomodulatory property of an acidic polysaccharide from mycelial culture of cordyceps sinensis fungus Cs-HK1. Food Chem 125(2):637–643 Wang X, Tao F, Gai Z, Tang H, Xu P (2012) Genome sequence of the welan gum producing strain Sphingomonas sp. ATCC 31555. J Bacteriol 194:5989–5990 Wasser SP (2010) Medicinal mushroom science: history, current status, future trends, and unsolved problems. Int J Med Mushrooms 12(1):1–16 Weng BBC, Lin YC, Hu CW (2011) Toxicological and immunomodulatory assessments of botryosphaeran (β-glucan) produced by Botryosphaeria rhodina RCYU 30101. Food Chem Toxicol 49(4):910–916 Wu SH, Nilsson HR, Chen CT, Yu SY, Hallenberg N (2010) The white rotting genus Phanerochaete is polyphyletic and distributed throughout the phleboid clade of the Polyporales (Basidiomycota). Fungal Divers 42:107–118 Wu S, Chen J, Pan S (2012a) Optimization of fermentation conditions for the production of pullulan by a new strain of Aureobasidium pullulans isolated from sea mud and its characterization. Carbohydr Polym 87(2):1696–1700 Wu X, Xu R, Ren Q, Bai J, Zhao J (2012b) Factors affecting extracellular and intracellular polysaccharide production in submerged cultivation of Tricholoma mongolicum. Afr J Microbiol Res 6(5):909–916 Xiao JH, Xiao DM, Xiong Q, Liang ZQ, Zhong JJ (2010) Nutritional requirements for the hyperproduction of bioactive exopolysaccharides by submerged fermentation of the edible medicinal fungus Cordyceps taii. Biochem Eng J 49:241–249 Yang H, He G (2008) Influence of nutritional conditions on exopolysaccharide production by submerged cultivation of the medicinal fungus Shiraia bambusicola. World J Microbiol Biotechnol 24(12):2903–2907 Yang FC, Ke YF, Kuo SS (2000) Effect of fatty acids on the mycelial growth and polysaccharide formation by Ganoderma lucidum in shake flask cultures. Enzyme Microbiol Tech 27 (3–5):295–301 Yang BK, Jeong SC, Park JB (2001) Swimming endurance capacity of mice after administration of exo-polymer produced from submerged mycelia culture of Ganoderma lucidum. J Microbiol Biotechnol 11(5):902–905 Yin Y, Hu Y, Xiong F (2011) Sorption of Cu(II) and Cd(II) by extracellular polymeric substances (EPS) from Aspergillus fumigatus. Int Biodeter Biodegr 65(7):1012–1018 Zhai X, Zhao A, Geng L, Xu C (2012) Fermentation characteristics and hypoglycemic activity of an exopolysaccharide produced by submerged culture of Stropharia rugosoannulata 2. Ann Microbiol 4(1):754–772

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Isolation and Purification of Microbial Exopolysaccharides and Their Industrial Application Veena S. More, Allwin Ebinesar, A. Prakruthi, P. Praveen, Aneesa Fasim, Archana Rao, Farhan Zameer, K. S. Anantharaju, and Sunil More

Abstract

Exopolysaccharide is long-chain high molecular weight polymeric carbohydrates composed of monosaccharide units bound together by glycosidic linkage, which are secreted extracellularly by the secreting microscopic cell or organism. These exopolysaccharides can be either homopolysaccharide or heteropolysaccharide in nature. Exopolysaccharide comprises repeated units of sugar moieties attached to the carrier lipid and can be associated with proteins, lipids, organic and inorganic compounds, and metal ions. Bacteria, archaea, yeast, filamentous fungi, and single cell of eukaryotes produce microbial exopolysaccharides. Microbial synthesis of polysaccharides is greatly influenced by environmental factors such as temperature, pH, pressure, salinity, toxicity, and radiation levels across their ecological niche. Due to the very less production time of exopolysaccharides and its simple purification process, these have found various successful applications in various industrial sectors such as pharmacology, diagnostics, nutraceuticals, functional foods, cosmetics, herbicides and insecticides, bioremediation, biotechnology, petrochemicals, dairy industry, and paint industry. Some of the most important exopolysaccharides and its industrial applications are Acetan (as preservative), Alternan (as commercial gum Arabic), Biodispersion (as remediation of oil spills), Cellulose (as non-indigestible fiber), Curdlan (as a food additive and gelling agent), Dextran (as moisturizers), Emulsan (as crude oil recoveree), Gellan (as stabilizer and microencapsulation matrix), Hyaluronan V. S. More (*) · A. Ebinesar · P. Praveen Department of Biotechnology, Sapthagiri College of Engineering, H. Ghatta, Bangalore, India A. Prakruthi · A. Fasim · A. Rao · F. Zameer · S. More School of Basic and Applied Sciences, Dayananda Sagar University, Bangalore, India K. S. Anantharaju Department of Chemistry, Dayananda Sagar College of Engineering, Bangalore, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_3

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(as for the removal of dead cells in skincare products), Kefiran (as anti-cancer agents), Levan (as viscosifier), Mutan (as adhesives), Succinoglucan (as emulsion stabilizers), Welan (as thickening agents in high-temperature industries), Xanthan (as an emulsifier and suspending agent), and Pullulan (as orally consumable films). These exopolysaccharides are produced by bacterial species such as Acetobacter, Pseudomonas, Leuconostoc, Alteromonas, Alcaligenes, Lactobacillus, Zymomonas, Xanthomonas, Aureobasidium (fungus). These compounds are purified by techniques such as membrane filtration, dialysis, precipitation, various types of column chromatography, lyophilization, distillation, and rotatory vaporization. This chapter describes different isolation and purification techniques for microbial exopolysaccharides. Keywords

Exopolysaccharide · Bacteria · Chromatography · Purification

3.1

Introduction

Microbes being one among the most abundant live forms in this biosphere are known to produce many extracellular metabolic products, commonly known as extracellular products or exopolysaccharides. Microbes are found in all forms of habitable or non-habitable ecological conditions. Microbes consist of domains such as archaea, mycota, and fungi. Microbial exopolysaccharides are found in extracellular space, long chain, and high molecular weight polymeric carbohydrates composed of monosaccharide units bound together by glycosidic linkage. These exopolysaccharides can be found in two forms, homopolysaccharide and heteropolysaccharide. Homopolysaccharides are those that consist of same repeating units of monomeric molecules and heteropolysaccharides are those that consist of different repeating units of monomeric compounds. Microorganisms are living in very harsh environmental conditions such as arid deserts, geographical poles, volcanoes, deep trench, rich ecological niche, acidic environments, and many more commonly synthesized exopolysaccharides. These exopolysaccharides are highly influenced by the factors of nature such as temperature, pH, humidity, toxicity, pressure, and radiation levels. Exopolysaccharides consist of repeated units of sugar moiety attached to the carrier lipid and can be found associated with proteins, lipids, organic or inorganic compounds, and metal ions. Microorganisms such as bacteria, archaea, yeast, filamentous fungi, and single cell of eukaryotes produce exopolysaccharides. Exopolysaccharides are usually found attached to the cell membrane of microbes, or as free molecules in the extracellular space. Exopolysaccharides are usually synthesized by microorganisms for their protection from pathogens and virulent organisms, as removal of endotoxins for the microorganisms, as by-products of metabolism, for symbiotic relationship with the host. As these are found in outer space of microorganisms, exopolysaccharides are very easy to isolate, purify, and find varied applications in industrial sector.

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In this chapter, we will discuss some of the most important and commonly used microbial exopolysaccharides such as alternan, cellulose, curdlan, dextran, gellan, hyaluronan, kefiran, levan, welan, xanthan gum, pullan, and also their isolation and purification techniques such as salting out method, ultracentrifugation method, ultrafiltration method, and various chromatography techniques such as anion exchange column chromatography, affinity, and chromatography. Industrial applications of above-mentioned various enzymes are also discussed.

3.2

Various Exopolysaccharides

3.2.1

Xanthan

Xanthan gum was first discovered in 1960s and in the late 1970s it was commercialized (Barcelos et al. 2020). Xanthan is produced by pure cultures of Xanthomonas campestris (Frese et al. 2014). Xanthan is a long-chain polysaccharide consisting of D-glucose, D-mannose, and D-glucuronic acid as building blocks in molecular ratio of 3:3:2 with high number of trisaccharide side chains. Average molecular weight of xanthan is about 2000 kDa (Jindal and Khattar 2018). Xanthan gum is a water-soluble fast hydrating hydrocolloid, which easily dissolves at room temperature. Stirring and continuous mixing increases the dissolution process of Xanthan gum. Generally, greater the ionic strength more is the dissolvation time. Xanthan solution can function well in the pH range of 3–10. Xanthan gum (XG) is recommended due to its high solubility in cold and hot water, stable viscosity at lower concentrations. To add on the helical conformation possessed by XG displays intense pseudoplastic performance and helps in instant recovery.

3.2.1.1 Pharmaceutical Applications XG is currently being used in pharmaceutical preparations due to its branched polymeric structure. The cohesive and adhesive characteristics is majorly because of the special structure it possesses. Additionally, it finds its application as a thickening, stabilizing, gelling, and emulsifying agent along with being used as tablet binder. Viscoelastic properties of xanthan solutions form a weak gel, giving characteristic suspending in liquids. Xanthan solutions are more stable than other thickeners and even resistant to enzymes, such as amylases, proteases, and cellulases (Wüstenberg 2014). A study was conducted to understand the encapsulations of β-carotene for stabilizing pickering emulsions of wheat gluten nanoparticles by using xanthan gum. It was established that the utilization of xanthan helped in preventing the chemical degradation of β-carotene during storage for 1 month. It could help in sustaining at higher pH and excessive salt concentration as well. In another study, XG was supplied to CMXG, and further cross-linked as a hydrophilic polymer with Ca+2 ion (in situ) for the research of prednisolone matrix tablets. The usage of XG assisted in the reduction of immediate release of the drug in designated regions like upper gastrointestinal tract.

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3.2.1.2 Personal Hygiene Products In products like toothpaste, shampoos, and liquid soap, XG assists in boosting the flow properties by this means enabling the easy extrusion from pump dispensers and tubes. The effect of XG displaying a pseudoplastic behavior also helps in offering an increased level of smoothness and softness in creams, lotions, and gels. Xanthan was employed in rice bran oil formulation for its utilization in sun protection agents. The concentration of XG was varied and tested to check its emulsification properties. The droplet size varied and the zeta potential values were augmented; however, the pH remained consistent throughout. It was thus concluded that XG can be used to enhance the physicochemical properties of the oil which can further be used in different fields. 3.2.1.3 Oil Industry The stability factor of XG makes it easily utilized in petroleum industries as an additive in cleaning pipes, drilling fluids, and in enhancing the recovery of oil. Hydrolyzed polyacrylamide has been replaced with xanthan in oil recovery process as it is inadequate to reservoirs with moderate or low hardness brine solution. It cannot be frequently employed even during lower temperatures as their viscosities get highly affected by the presence of electrolytes. This is possible due to its tolerance towards high salt concentration and the capacity to maintain viscosity of fluids that are water based. XG is also known to be used in micellar-polymer flooding in the recovery of oils. The oils that are trapped in tiny gaps of small sandstone need to be pumped by using polymer-based salt solutions. XG helps in easy recovery of residual oil and helps in maintaining the efficiency of oil extraction. 3.2.1.4 Food Processing Industries Plastic materials which are used in food packaging are being replaced by polysaccharides like xanthan due to its contribution in increasing the film forming capacity due to its stability in hot and cold water. It acts as an excellent UV light shielding component along with maintaining the moister content of the food. (Abu Elella et al. 2020). 3.2.1.5 Other Applications Another interesting contribution of XG is in the field of catalytic application, due to its active functional groups which include carboxyl and hydroxyl moieties. The presence of these groups increases the dispersal property of catalytic nanoparticles such as Pd, Ag, and aid in the decreased size for enhancing their catalytic accomplishment. Reports also explain XG being used in the treatment of contaminated water. XG-based hydrogels and nanocomposites are used as they exhibit high surface area (specific), gelation capabilities, and thermal stability along with great mechanical strength properties. Thus, xanthan can be used as an alternative in the above-mentioned fields and exploratory research of its other applications is yet to be investigated.

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Gellan

Gellan gum is exopolysaccharide produced by Sphingomonas elodea by fermentation (Bajaj et al. 2007; Wang et al. 2006). Gellan is linear, anionic heteropolysaccharide, straight chain consisting building blocks of D-glucose, Lrhamnose, and D-glucuronic acid in molecular ratio of 1.5:1:1. The chain consists of tetrasaccharide repeating unit in which beta (1–4) linked glucose, glucuronic acid, glucose, and rhamnose in alpha (1–3) linkage are bonded together. The native product is partially esterified. In its native or high-acyl form, two acyl substituents acetate and glycerate are present. One glycerate for one repeat and one acetate for every two repeating units are observed. Gellan is suspected to adopt two- or threefold helical structure after heating or cooling (Ferreira et al. 2016). Gellan gum easily dissolves in hot and cold water. In high gellan gum concentration in solutions, a network-like structure is observed called demoldable gel. In lower gellan gum concentration in solutions, molecules are associated closely but system remains fluid, hence known as fluid gel (Nitta and Nishinari 2005; Coviello et al. 2007).

3.2.2.1 Pharmaceutical Applications Gellan gum is used in biomedical and pharmaceuticals research as a protein carrier. It also finds its application in gene transfection and gene therapy. Gellan gum combined with chitosan was effectively used in the manufacture of nasal insert which aided in the enhancement of drug release and water up-taking capacity. The combinational product was a replacement to other polymers which displayed deficiencies with their efficacy. The drug release at lower pH is not possible by compounds like chitosan along, hence when polysaccharides like gellan are used along. The facilitation of drug release is improved. Nasal cavity is handled mostly for the local treatment of nasal infections and congestion. It was studied that gellan amended the epithelial transport further successfully than the usually employed isotonic D-mannitol solution. Furthermore, the dwelling time in the nasal cavity was up to 4 h and there was no side effects noted (Osmałek et al. 2014). 3.2.2.2 Tissue Engineering Gellan gum has been lately examined in the arena of tissue engineering, as a substantial for cartilage restoration. The ability to self-repair after a damage to tissue is very inadequate. Hence, it is important to note that the ability of cartilage to selfrepair after degeneration or mechanical damage is limited. Therefore, biomaterials to repair damaged tissue is essential. Gellan-based structures are presently discovered as injectable carriers for numerous autologous cells like bone marrow cells and chondrocytes. In vivo results of viability and proliferation were promising when conducted (Osmałek et al. 2014).

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3.2.2.3 Food Industry A study reported that when gellan was combined with pectin (methoxy pectin) the aroma compounds present in food products, wine, and essential oils helped in retaining the aroma for an extended duration. Also the same combinational product was utilized in the preparation of dough, a nutritious acidic milk prepared from yogurt. After the addition, it was noted that the usability of the product was improved by increasing the stability (Zia et al. 2018).

3.2.3

Pullan

Pullan being an extracellular glucan by fungal strain Aureobasidium pullulans commonly known as black yeast through fermentation (Choi et al. 2014). Pullan is linear homopolysaccharide consisting of maltotriose as monomers. Three glucose units of maltotriose are linked through alpha (1–4) glycosidic linkage, and maltotriose units are linked through alpha (1–6) linkage. Molecular weight of pullan is between 10 and 400 kDa (Mano et al. 2007; Navard 2012). Pullan forms a stable, viscous fluid when dissolved in hot or cold water but does not form a gel (Imeson 2011). Pullan is insoluble in organic solvents except dimethyl sulfoxide and dimethylformamide. Viscosity of pullan remains not much affected in the pH range of 2–11. In addition, it remains stable in the presence of ions (Cui 2005). Pullan is an odorless and tasteless white powder. Pullan is heat resistant and it readily forms film, which is oil repellent, transparent, edible, and readily soluble in water (Shit and Shah 2014).

3.2.3.1 Food Processing Industries Due to its non-toxic, non-immunological, and non-carcinogenic properties pullan is extensively used in food edible coatings, as foaming agents and flocculants (Zia et al. 2018). Edible coatings assist in retarding dehydration which in turn recover the quality of the food medium by retaining the volatile flavor complexes and also decrease bacterial growth. Pullan-based films can interact with the food by enriching the safety and quality by releasing essential compounds. Pullan is used for preparation of coating along with whey protein. Chestnuts were dipped in these film-forming solutions to reduce the crop wastage because of high water wastage. SEM results were conducted to check the adherence of the coated films and the results conveyed the active properties of pullan-based coated films. Further experiments also demonstrated that moisture loss was reduced and the shelf life of the chestnuts were improved (Trinetta and Cutter 2016).

3.2.4

Dextran

Dextran is an extracellular product of Streptococcus or Leuconostoc by fermentation of sucrose at optimum pH 6.5–7 and optimum temperature of 25–30
C. The

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commercial dextran is produced by L. mesenteroides and L. dextranicum (Wüstenberg 2014). Dextran is synthesized using dextransucrase in two pathways. • By hydrolyzing the sucrose and binding the glycosyl moiety. • Building up the dextran by the insertion mechanism. Dextran is highly branched, neutral, high molecular weight homopolysaccharide made up of glucose monomers. Glucose monomers are linked together through alpha (1–4) linkage. Dextrans have 10–50,000 kDa molecular weight (Kapoor et al. 2013). Dextran is readily soluble in water or glycerin at room temperature. Dextran solution is highly viscous or slimy fluid. Dextran size and structure greatly depends on dextransucrase enzyme produced by the different microbial strains. This significantly justifies the difference in the structure of dextran isolated from diverse bacterial species. Dextran prevents crystallization, improves moisture retention, and increases viscosity in the solution. Dextrans are odorless, tasteless, and non-toxic, and these characteristics are favorable over other stabilizers. Humans can easily digest dextran, and hence it serves as high-energy source (Bhavani and Nisha 2010; Kothari et al. 2014).

3.2.5

Curdlan

Curdlan is a secondary extracellular polysaccharide high molecular weight polymer of glucose produced by a non-pathogenic bacterium Agrobacterium biobar and mutants of Alcaligenes faecalis (Zhang and Edgar 2014). It is a linear beta (1–3) glucan. It is partially esterified with succinic acid and appears as succinoglucan (Wüstenberg 2014). Curdlan dissolves in aqueous alkaline solution and is an insoluble exopolysaccharide in water and alcohol. Curdlan is known to form strong thermos irreversible high set elastic gel at temperatures above 54
C. Gel strength is known to be between agar and gelatin (Saha and Bhattacharya 2010). Curdlan when heated with solvent forms an irreversible gel. Gel strength increases with an increase in temperature. Curdlan forms water-insoluble films. These gels are known to be biodegradable, impermeable to oxygen, and edible. Curdlan is an odorless white powder (Ghanbarzadeh and Almasi 2013).

3.2.5.1 Food Processing Industry Curdlan displays thermal gelling and finds its applications in various food industries and is legally permitted to be used as a food additive in countries like the USA, Japan, Taiwan, and Korea. Curdlan comprises dietary fibers which are highly insoluble and are thus used in the production of low-calorie foods. It is likewise used as a stabilizer, texturizer in jelly-based food and in meat products, dairy products, respectively. Potato starch and other starch are extensively used in the noodle industry due to the need of higher

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content of amylose. Addition of curdlan improved the viscidness and solidity along with the gradual decrease in the stickiness of the noodles (Verma et al. 2020).

3.2.5.2 Biomedical Applications Curdlan also illustrates potential applications in the field of biomedical and pharmaceuticals. Gel encapsulation of indomethacin, prednisolone was performed using curdlan to check for better drug release. When the drug release system was compared and analyzed, curdlan-gels were found to remain intact and improved the drug release location. This also helped in avoiding drug uptake into portal vein (Zhang and Edgar 2014).

3.2.6

Levan

Levan is a fructose-based polysaccharide, which is extracellularly secreted by many microorganism species. Some of the microorganisms are Acetobacter, Bacillus, Brenneria, Geobacillus, Halomonas, Lactobacillus, Zymomonas, and Saccharomyces (Han 1990). Levan is composed of beta D-fructofuranose with beta (2–6) linkages between fructose rings. Levansucrase synthesizes levan by directly converting sucrose into a levan polymer. Levan is highly soluble in water and oils, however is insoluble in many organic solvents such as methanol, ethanol, and isopropanol. Large amount of tensile strength and cohesiveness of levan is due to its branching state of existence. Adhesiveness with molecules is achieved by the presence of hydroxyl groups. Levan has a very low viscosity and is non-toxic in nature. Levan has high value of adhesivity, strong biocompatibility, and film forming ability (Ullrich 2009).

3.2.6.1 Tissue Engineering Reports discuss the application of levan as a bioadhesive agent in drug delivery systems. In tissue engineering, levan is successfully used as a material for scaffold preparation as a remedy for wound healing. 3.2.6.2 Biotechnological Applications Due to its adhesive nature and water retracting properties, levan is advantageous in assisting microbes and plants to survive in regions with limited source of water. Thereby increasing the water availability in hypersaline environments as well which forms the major portion of earth ecosystem. 3.2.6.3 Other Applications In the manufacturing of adhesive membranes, film casting a conventional technique. In the process, levan from microbial sources were supplemented with chitosan and polyethylene (PEO) as a casting solution. Levan facilitated an improvement with the biocompatibility of the films. Furthermore, mechanical and thermal characteristics were maintained by suppressing the crystallinity of PEO.

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It is an ideal biopolymer for many industrial applications due to its wide range of potential characteristics. (Combie and Öner 2018).

3.2.7

Welan

Welan is a novel fermented exopolysaccharide product of Sphingomomonas species, a Gram-negative bacterium and by Alcaligenes species, a mutant species. Of the two species, Alcaligenes species is known to produce welan in a large amount of quantity (Kaur et al. 2014). Welan is an acidic heteropolysaccharide and has an approximate molecular weight of 1.0*10^6 g/mol (Kaur et al. 2014). The extracellular polysaccharide welan gum is a series of oligosaccharides, which consists of L-mannose, Lrhamnose, D-glucose, and D-glucuronic acid as backbone in the molar ratio of 1.0:4.5:3.1:2.3. Welan has a single side chain containing either L-rhamnose units or L-mannose units substituted on C3 of every 1,4 glucose repeating units (O’Neill et al. 1986). The presence of side chains greatly reduce the gelling behavior of the welan. Welan in aqueous solutions has a weak gel property. Welan gum polymer produces a high viscous aqueous solution exhibiting higher viscosity retention at high temperature (up to 150
C) and wide pH range of (2–12) (Jansson et al. 1985).

3.2.7.1 Cement Industries The utilization of welan gum in cement configurations has a diverse implication. It decreases fluid loss of the cement compositions and upsurges the suspension characteristics of cement. Also is highly operative in low concentration as well. It is widely used in oilfield processes which include wellbore cleanup, hydraulic fracturing, and drilling for viscosity enhancement. It has its application in oil well drilling as a spacer fluid. 3.2.7.2 Other Applications Welan gum has certain qualities like binding, thickening, and also display emulsifying properties. Hence, it finds its application as an constituent in beverages like citric acid centered drinks and dairy food products like yoghurt and ice creams (Kaur et al. 2014). Oil recovery is one such field where welan is used because of its gelling properties. The decent thermal insulating characteristics and rheology of the gel make them feasible as an insulating material for pipeline bundles so that the temperature (above 30
C) is maintained and is above sea level.

3.2.8

Kefiran

Kefiran is a capsular polysaccharide synthesized extracellularly by slime forming rod-shaped Lactobacillus species. Kefiran is a fermented product obtained from kefir grains (Farnworth 2006). Kefiran is soluble in water and in aqueous solutions containing ethanol forms gels. Branched glucogalactan comprises equal amount of

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D-glucose

and D-galactose residues. Kefiran is resistant to hydrolysis. Kefiran has a structure of hexasaccharide or heptasaccharide repeating units which itself composed of pentasaccharide units to which one or two residues are randomly linked. Primary structure of kefiran is known to consist of galactose and glucose in the molar ratio of 1.1:0.9.

3.2.8.1 Food Industry Kefiran has been reported to possess antibacterial, antifungal, and antitumor properties (Rodrigues et al. 2005). Kefiran has been shown to enhance the production of beta cortisol and noradrenaline in human cell lines. It also finds its application in food industry (Agoub et al. 2007). Studies have reported that kefiran combined with whey protein isolate was used to manufacture bio-nanocomposites with nanofillers which exhibited greater efficiency in acting as oxygen and moisture barriers. Hence, they can be used in food packaging industries. Recent studies also suggest that when kefiran was added to the films for food packaging, it also assisted in the acting as a UV-blocking. UV-blocking is a very vital factor in edible food packaging sector. Kefiran is considered to be a stress-reducing food supplement due to its competence in increasing the production of interferon B-cortisol and noradrenaline in human cell lines. 3.2.8.2 Medical Applications Research was conducted to determine and demonstrate the efficiency of kefiran in treating Candida albicans by improving the protective outcome to dermal connective tissue and encouraging wound healing. This study was conducted in vivo to justify the outcome. In human intestinal system immune system, kefiranis found to act as a stimulant by altering the immune cells (Tan et al. 2020).

3.2.9

Hyaluronan

Hyaluronan also known as hyaluronic acid is a linear high molecular weight heteropolysaccharide consisting of alternate beta 1, 3 N-acetylglucosamine and beta 1, 4-glucuronic acid linked through beta-(1–4) and beta-(1–3) glycosidic bonds (Chen et al. 2014a, b). Hyaluronan is synthesized by the bacterial species of streptococcus such as S.equisimilis, S.pyogenes, S.thermophilus, and S.equi (Shiedlin et al. 2004). A recombinant strain of Bacillus subtilis is known to produce hyaluronan at laboratory scale. Hyaluronan can be approximately 20,000–25,000 disaccharide repeats in length (Chen et al. 2014a, b). Hyaluronan has a very high rate of water holding capacity, high biocompatibility, and high viscosity. Hence, it is considered as a natural lubricant. It is energetically stable, in part because of the stereochemistry of its component disaccharides. Bulky groups on each sugar molecule are in sterically favored position; likewise, the small hydrogen groups assume the less favorable axial position (Izawa et al. 2009, 2011). Due to these various properties of hyaluronan, it finds applications in various industries which include in the manufacture of ophthalmic products, treatment of ulcers and surgical procedures.

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3.2.9.1 Medical Applications Hyaluronan plays a dynamic role in the growth of cartilage and in the sinovial fluid maintenance for tendon regeneration. Hyaluronan is thus present in greater concentrations in the ECM of all adult joint tissues. Thus, HA acts in the joint as a lubricant and shock absorber and can externally supplied to improve bone health. Study reports that hyaluronan hydrogels have accelerated the healing of wounds by providing highly hydrated non-immunogenic environment that assisted in faster healing process. Hyaluronan has been extensively used in the field of cosmetics due to its property of hydration. Hyaluronan has also proven to be operative for intensifying the blood compatibilities of cardiovascular implants such as stents and vascular grafts. Biomaterial surfaces treated with cross-linked hyaluronan have been associated with reduced platelet adhesion and thrombus formation. HA derivatives with higher degrees of sulfation are connected with augmented aptitudes to avoid blood coagulation (Necas et al. 2008)

3.2.10 Alternan Alternan is a microbial high molecular weight exopolysaccharide consisting of alternating sequence of alpha (1–3) and alpha (1–6) linked D-glucose units (Misaki et al. 1980). Some single extracellular enzymes of certain strains of lactic acid bacterium Leuconostoc mesenteroides produce alternan. Alternan are synthesized as extracellular slimes by lactic acid bacteria (Cote et al. 2002). Due to an unusual linkage structure, alternan possesses resistance to many hydrolytic enzymes that degrade starch, dextran, and other alpha D-glucan. Alternan possesses low viscosity than those posed by other food gums. Alternan is readily soluble in water (Leathers et al. 2003). Due to similarity of alternan in structure with dextran, alternan finds applications in the artificial sweeteners in the sugar confectionary industries.

3.2.11 Cellulose Cellulose is an exopolysaccharide synthesized by various species of bacteria such as Gluconacetobacter, Agrobacterium, Rhizobium, Salmonella, and Sarcina. Among these, Gluconacetobacter is considered as the most cellulose producing species (Glenn et al. 2009). Cellulose is a polysaccharide composed of carbon, hydrogen, and oxygen. Cellulose is a straight chain polymer whose base units of glucose beta linkages hold together. Each polymeric chain is asymmetric containing reducing end and non-reducing end. The reducing end has carbonyl functional group whereas non-reducing end has hydroxyl functional group (de Souza Lima and Borsali 2004). Cellulose are found in different physical states such as crystalline, amorphous, sheets, and fibers. Cellulose fibrils are insoluble, inelastic, and have tensile strength comparable to that of steel. They possess high mechanical strength, water absorption capacity, temperatures, crystallinity, pure fine fiber network, and are highly stable to

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chemicals. Cellulose sheets are known to be resistant to higher stability of pressure (Glenn et al. 2009; Hokkanen et al. 2016). Cellulose is tasteless, odorless, hydrophobic, chiral, renewable, biodegradable, and is insoluble in water and organic solvents. Due to all these varied properties, cellulose finds applications in medicinal, food, commercial, and industrial fields.

3.2.11.1 Food Industry Cellulose nanocrystals (CNC) are used to enhance the mechanical characteristics of polymers which include soy protein, rubber latex, and starch-based matrixes. They find their applications in food packaging industries as coating materials. They act as excellent oxygen barriers and also enhance the biodegradable properties. In specific, nanocellulose can be used as a low calorie substitute for carbohydrate extracts as thickeners, flavor carriers, and suspension stabilizers in a wide variety of food products. It is useful for producing crushes, fillings, chips, soups, wafers, gravies, etc.

3.3

Isolation and Purification Techniques

For microbial exopolysaccharides, the various downstream processes of isolation and purification do not have a clear distinguishing difference because sometimes the isolation process is a part of purification process itself and vice versa. Hence, in this chapter we discuss some of the most important and common techniques of purification and isolation of the microbial exopolysaccharide.

3.3.1

Ultracentrifugation Method

Ultracentrifugation method is one of analytical techniques, which involves ultracentrifuge with optical monitoring systems. Various polysaccharides possess different molecular weight and different sedimentation speed in strong centrifugal force. Using this property, various polysaccharides can be purified. Ultracentrifugation is of two types, one is differential centrifugation and other is density gradient zonal centrifugation method (Shi 2016). Different molecular weight polysaccharides are separated in batches by gradual addition of speed in the differential centrifugation method. The high molecular weight polysaccharides are separated in the low speed whereas the low molecular weight polysaccharides are separated at higher speeds. Density gradient zonal centrifugation is used to detect the homogeneity of the polysaccharides. The rationale of this method is when polysaccharides are centrifuged in the inert gradient media and reach equilibrium. Various polysaccharides can be collected and distributed to certain specific positions within gradient and form different zones. These zones are separated and different

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polysaccharide fractions are obtained. Commonly used inert media are water, NaCl, CsCl solution, and many more (Shi 2016).

3.3.2

Ultrafiltration Method

Ultrafiltration works on the principle of pressure-induced separation of solutes from solvent through semi-permeable membrane. The relationship between the applied pressure on the solution to be separated and the flux through the membrane is most commonly described by Darcy equation. Suspended solids and solutes of high molecular weight are retained in retentate, while water and low molecular weight exopolysaccharides pass through the membrane in permeate or filtrate. Ultrafiltration principle is also defined as molecular sieve, which mainly works on size exclusion or particle capture mechanism in the membrane. Ultrafiltration membranes are defined by molecular weight cut-off value of the membrane used. Ultrafiltration is applied in cross flow or dead end mode (Schachman 2013).

3.3.3

Salting Out Method

Salting out is also known as salt-induced precipitation, salt fractionation, antisolvent crystallization, precipitation crystallization, or drowning out. Salting out method is based on the electrolyte–nonelectrolyte interaction, in which the non-electrolyte could be less soluble at high concentration. This method is used for purification of protein and to prevent protein denaturation due to excessively diluted samples. Salt compounds dissociate in the aqueous solutions. When the salt concentration is increased, some of the water molecules are attracted by the salt ions, which decreases the number of water molecules available for the interaction with the charged part of exopolysaccharides. The principle of this method is that different molecular weight polysaccharides have different solubility in the salt solutions of certain concentrations. When neutral salt is added to the polysaccharide solution and when it reaches equilibrium with the solution, the exopolysaccharide precipitates out from the solution. This precipitate can be separated by centrifugation.

3.3.4

Anion Exchange Column Chromatography

Anion exchange column chromatography is the widely used analytical technique for polysaccharide isolation and purification. It is used for bulk quantity isolation of polysaccharide. Concentration and preliminary purification of polysaccharide solution is possible in this method. The most widely used anion exchanger so far is DEAE-cellulose, DEAE-sephadex, and DEAE-sepharose. The separation mechanism of anion exchange column chromatography is ion exchange and adsorptiondesorption. Hence, anion chromatography can be used in separation of neutral

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polysaccharide, acidic polysaccharide, and mucopolysaccharide. Acidic polysaccharide is adsorbed to the exchanger whereas neutral polysaccharides cannot be absorbed. Then the buffers, which have the same pH value but different values, can be used to elute these acidic polysaccharides, respectively. The adsorption ability generally increases with an increase in acidic groups in the polysaccharide; the adsorption ability of straight chain polysaccharide is greater than branched chain polysaccharide. The height of column bed and flow rate of the polysaccharide also greatly affects this method of isolation and purification (Sober et al. 1956).

3.3.5

Affinity Chromatography

Affinity is generally known as the binding ability between two different molecules. Two specific molecules can dissociate after they bind. Using this principle, polysaccharides can be isolated, purified by the process of binding-dissociation. The affinity column is eluted using polysaccharide solution as mobile phase. As polysaccharide solution is a mixture of multiple polysaccharides, only specific polysaccharide will bind to the affinity column and are adsorbed whereas the remaining polysaccharide will elute from the column. The ionic strength and pH value of the mobile phase can be changed to dissociate the polysaccharide fraction combined with ligand. Hence, purified polysaccharide fraction can be obtained. The advantage of the affinity chromatography is that it has high efficiency and is easy to operate. The most important aspect and advantage of affinity chromatography usage that is several hundred to several thousand fold of concentration purity can be achieved (Cuatrecasas and Anfinsen 1971).

3.4

Industrial Applications of Exopolysaccharides

Polysaccharide Xanthan

Gellan

Pullan

Industrial applications Emulsification and gelationation in food industry, thickener, suspension stabilizer in pharmaceuticals creams, oil recovery, mineral ore processing, paper manufacturing, agriculture, cosmetics, ice creams, puddings, cheese spreads, toothpaste, textile, and agriculture Solidification agent in beverage industry, excipient in oral, ophthalmic, and nasal drug formulations, tablet dis-integration, jams, jellies, microencapsulation Tablet granulation and coating, binder, and oxygen impermeable film forming, non-animal capsules, oral, and wound care products, food

References Born et al. (2002), Morris and Hardling (2009), Saha and Bhattacharya (2010)

Felt et al. (2002), Hagerstrom (2003), Osmałek et al. (2014), Mariod and Fadul (2013)), Saha and Bhattacharya (2010)) Ferreira et al. (2016), Shit and Shah (2014)

(continued)

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Dextran

Curdlan Levan

Welan

Kefiran Hyaluronan

Alternan Cellulose

3.5

additive in low calorie foods, preservative, food packing industry Blood plasma extender, cholesterollowering agent, microcarrier in tissue and cell culture techniques, artificial sugars Gelling agent, ice creams, yogurts, cheese, salads, preservatives Blood plasma extender, cholesterollowering agent, prebiotic, fodder for animals Stabilizer and viscosifier in food industry, jellies, beverages, dairy products, cosmetics, capsule coating of tablets Gelatination and viscoelasticizer in dairy industry Moisturizers in skin care industry, synovial fluid substitute in stem cell industry, chronic difficult wound healing, eye surgery, disinfectant Blood flow improving agent, cholesterol-lowering agent Natural non-indigestible fiber, temporary artificial skin, acoustic membranes in audiovisual equipment

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Nwodo et al. (2012), Bhavani and Nisha (2010), Kothari et al. (2014)

Chien and Iwata (2018) Patel and Prajapati (2013), Kang et al. (2009) Asubiaro and Shah (2008), Ma et al. (2012)

Kogan et al. (2006), Necas et al. (2008)

Fang and Catchmark (2015), Chawla et al. (2008)

Conclusion

As evident from the review of the numerous EPS produced from the different microorganisms remains in the chapter remains to be an unexplored field of study which finds it applications in industries. The eco-friendly exopolysaccharides discussed here are mainly carbohydrates and determines their functions in respective fields. The various isolation and purification techniques mentioned here can be optimized and used in large-scale production of EPS due to its extensive application purposes. The outline of the chapter mainly holds to initiate a proper characterization and documentation of EPS composition and functionality to understand its applications in distinguished fields.

References Abu Elella MH, Goda ES, Gab-Allah MA, Hong SE, Pandit B, Lee S, Gamal H, ur Rehman A, Yoon KR, 2020 Xanthan gum-derived materials for applications in environment and eco-friendly materials: a review. J Environ Chem Eng, 104702. https://doi.org/10.1016/j.jece. 2020.104702

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Agoub AA, Smith AM, Giannouli P, Richardson RK, Morris ER (2007) “Melt-in-the-mouth” gels from mixtures of xanthan and konjac glucomannan under acidic conditions: A rheological and calorimetric study of the mechanism of synergistic gelation. Carbohydr Polym 69(4):713–724 Asubiaro A, Shah SN (2008) Rheological and hydraulic properties of welan gum fluids in straight and coiled tubings. J Fluids Eng 130:081506 Bajaj IB, Survase SA, Saudagar PS, Singhal RS (2007) Gellan gum: fermentative production, downstream processing and applications. Food Technol Biotechnol 45(4):341–354 Barcelos MC, Vespermann KA, Pelissari FM, Molina G (2020) Current status of biotechnological production and applications of microbial exopolysaccharides. Crit Rev Food Sci Nutr 60 (9):1475–1495 Bhavani AL, Nisha J (2010) Dextran—the polysaccharide with versatile uses. Int J Pharm Biol Sci 1 (4):569–573 Chawla P, Bajaj IB, Survase S, Singhal RS (2008) Microbial cellulose: fermentative production and application. Biotechnology 47:107–124 Chen X, Gao H, Ploehn HJ (2014a) Montmorillonite–levan nanocomposites with improved thermal and mechanical properties. Carbohydr Polym 101:565–573 Chen X, Siu KC, Cheung YC, Wu JY (2014b) Structure and properties of a (1!3)-β-d-glucan from ultrasound-degraded exopolysaccharides of a medicinal fungus. Carbohydr Polym 106:270–275 Chien C-Y, Iwata T (2018) Synthesis and characterization of regioselectively substituted curdlan hetero esters with different ester groups on primary and secondary hydroxyl groups. Carbohydr Polym 181:300–306 Choi S, Choi W, Kim S, Lee SY, Noh I, Kim CW (2014) Purification and biocompatibility of fermented hyaluronic acid for its applications to biomaterials. Biomater Res 18(1):6 Combie J, Öner ET (2018) From healing wounds to resorbable electronics, levan can fill bioadhesive roles in scores of markets. Bioinspir Biomim 14:011001. https://doi.org/10.1088/ 1748-3190/aaed92 Cote Gl, Alternan Vandamme EJ, De baets S, Steinbuchel A (eds) (2002) Biopolymers, Wiley-vch, weinheim, chapter 13. P. 323–350 Coviello T, Matricardi P, Marianecci C, Alhaique F (2007) Polysaccharide hydrogels for modified release formulations. J Control Release 119(1):5–24 Cuatrecasas P, Anfinsen CB (1971) Affinity chromatography. In: Methods in enzymology, vol 22. Academic, Cambridge, pp 345–378 Cui SW (ed) (2005) Food carbohydrates: chemistry, physical properties, and applications. CRC Press, Boca Raton de Souza Lima MM, Borsali R (2004) Rodlike cellulose microcrystals: structure, properties, and applications. Macromol Rapid Commun 25(7):771–787 Fang L, Catchmark JM (2015) Characterization of cellulose and other exopolysaccharides produced from Gluconacetobacter strains. Carbohydr Polym 115:663–669 Farnworth ER (2006) Kefir–a complex probiotic. Food Sci Technol Bull 2(1):1–17 Ferreira AR, Alves VD, Coelhoso IM (2016) Polysaccharide-based membranes in food packaging applications. Membranes 6(2):22 Frese M, Schatschneider S, Voss J, Vorhölter FJ, Niehaus K (2014) Characterization of the pyrophosphate-dependent 6-phosphofructokinase from Xanthomonas campestris pv. Campestris. Arch Biochem Biophys 546:53–63 Ghanbarzadeh B, Almasi H (2013) Boidegradable polymers. In: Chamy R, Rosenkranz F (eds) Biodegradation- life of science. InTech Publications, Croatia, pp 141–186 Glenn GM, Holtman KM, Ludvik C, Chiou BS, Imam SH, Orts WJ, Wood D (2009) Green composites derived from natural fibers. In: Natural fibre reinforced polymer composites: from macro to nanoscale Han YW (1990) Microbial levan. In: Advances in applied microbiology, vol 35. Academic, Cambridge, pp 171–194 Hokkanen S, Bhatnagar A, Sillanpää M (2016) A review on modification methods to cellulosebased adsorbents to improve adsorption capacity. Water Res 91:156–173

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Imeson A (ed) (2011) Food stabilisers, thickeners and gelling agents. Wiley, Hoboken Izawa N, Hanamizu T, Iizuka R, Sone T, Mizukoshi H, Kimura K, Chiba K (2009) Streptococcus thermophilus produces exopolysaccharides including hyaluronic acid. J Biosci Bioeng 107 (2):119–123 Izawa N, Serata M, Sone T, Omasa T, Ohtake H (2011) Hyaluronic acid production by recombinant Streptococcus thermophilus. J Biosci Bioeng 111(6):665–670 Jansson PE, Lindberg B, Widmalm G, Sandford PA (1985) Structural studies of an extracellular polysaccharide (S-130) elaborated by Alcaligenes ATCC 31555. Carbohydr Res 139:217–223 Jindal N, Khattar JS (2018) Microbial polysaccharides in food industry. In: Biopolymers for food design. Academic, Cambridge, pp 95–123 Kang, S. A., Jang, K. H., Seo, J. W., Kim, K. H., Kim, Y. H., Rairakhwada, D., ... & Rhee, S. K. (2009). Levan: applications and perspectives. In: Microbial production of biopolymers and polymer precursors, pp 145–162 Kapoor M, Khandal RK, Seshadri G, Aggarwal S, Kumar Khandal R (2013) Novel hydrocolloids: preparation and applications–a review. IJRRAS 16(3):432–482 Kaur V, Bera MB, Panesar PS, Kumar H, Kennedy JF (2014) Welan gum: microbial production, characterization, and applications. Int J Biol Macromol 65:454–461 Kogan G, Šoltés L, Stern R, Gemeiner P (2006) Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications. Biotechnol Lett 29:17–25 Kothari D, Das D, Patel S, Goyal A (2014) Dextran and food application. In: Polysaccharides. Springer Leathers TD, Nunnally MS, Ahlgren JA, Côté GL (2003) Characterization of a novel modified alternan. Carbohydr Polym 54(1):107–113 Ma L, Zhao Q, Yao C, Zhou M (2012) Impact of welan gum on tricalcium aluminate–gypsum hydration. Mater Charact 64:88–95 Mano JF, Silva GA, Azevedo HS, Malafaya PB, Sousa RA, Silva SS et al (2007) Natural origin biodegradable systems in tissue engineering and regenerative medicine: present status and some moving trends. J R Soc Interface 4(17):999–1030 Mariod AA, Fadul H (2013) Gelatin, source, extraction and industrial applications. Acta Scientiarum Polonorum Technologia Alimentaria 12(2):135–147 Misaki A, Torii M, Sawai T, Goldstein IJ (1980) Structure of the dextran of Leuconostoc mesenteroides B-1355. Carbohydr Res 84(2):273–285 Navard P (ed) (2012) The European Polysaccharide Network of Excellence (EPNOE): research initiatives and results. Springer Necas J, Bartosikova L, Brauner P, Kolar J (2008) Hyaluronic acid (hyaluronan): a review. Veterinarni Medicina 53(8):397–411 Nitta Y, Nishinari K (2005) Gelation and gel properties of polysaccharides gellan gum and tamarind xyloglucan. J Biol Macromol 5(3):47–52 Nwodo U, Green E, Okoh A (2012) Bacterial exopolysaccharides: functionality and prospects. IJMS 13:14002–14015. https://doi.org/10.3390/ijms131114002 O’Neill MA, Selvendran RR, Morris VJ, Eagles J (1986) Structure of the extracellular polysaccharide produced by the bacterium Alcaligenes (ATCC 31555) species. Carbohydr Res 147 (2):295–313 Osmałek T, Froelich A, Tasarek S (2014) Application of gellan gum in pharmacy and medicine. Int J Pharm 466:328–340. https://doi.org/10.1016/j.ijpharm.2014.03.038 Patel A, Prajapati J (2013) Food and health applications of exopolysaccharides produced by lactic acid bacteria. Adv Dairy Res 1:1–7 Rodrigues KL, Caputo LRG, Carvalho JCT, Evangelista J, Schneedorf JM (2005) Antimicrobial and healing activity of kefir and kefiran extract. Int J Antimicrob Agents 25(5):404–408 Saha D, Bhattacharya S (2010) Hydrocolloids as thickening and gelling agents in food: a critical review. J Food Sci Technol 47(6):587–597 Schachman HK (2013) Ultracentrifugation in biochemistry. Elsevier

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A Review on Properties and Applications of Xanthan Gum Surabhi Chaturvedi, Sanchita Kulshrestha, Khushboo Bhardwaj, and Rekha Jangir

Abstract

Xanthan gum is a hetero-polysaccharide formed by the strains of Xanthomonas spp. It is a naturally found polysaccharide molecule of high molecular weight maily formed by various fermentation processes. Its extraordinary rheological properties make it a very useful stabilizing agent for water-based systems. It has enormous applications ranging from the food industry to oil drilling. It is typically used in food industry in salad coverings, sauces, milk products, gravies, sweets, and low calorie foods in general. Xanthan gum is also used in making cleansers, varnishes, polishes, and in agricultural flowables. This chapter describes the extraction procedure of xanthan gum from microbes, factors affecting production, and application in different sectors. Keywords

Xanthan gum · Oil drilling · Bakery · Food industry

S. Chaturvedi (*) · S. Kulshrestha · R. Jangir Lab No. 10, Department of Botany, University of Rajasthan, Jaipur, Rajasthan, India K. Bhardwaj Lab of Lifesciences, JECRC University, Jaipur, Rajasthan, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_4

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4.1

Introduction

4.1.1

History

In 1950, it was first learnt about xanthan gum (XG) at the National Center for Agricultural Utilization of the United Sates Department of Agriculture (USDA). Its first production by industries started in 1960. After dextran, it was the second polysaccharide, which was isolated from microbes and was adapted for industrial commercialization (Palaniraj and Jayaraman 2011). Xanthan gum is a heteropolysaccharide produced by the capsules of strains of Xanthomonas spp.which is usually associated with a loose bond with the bacterial cells which provides the best substitute of gluten. Its primary structure has repeats of pentasaccharide units containing two glucose, two mannose, and one glucuronic acid units with main chain comprising of β-d-glucose units interconnected at the first and fourth positions (Sworn 2009; Sharma et al. 2014). Galactomannan combinations made with xanthan gum are main ingredients to prepare gellies or thickeners of foods. It is also used as stabilizer in cattle-feed products, herbicides, fungicides, pesticides, fertilizers, and in toothpastes (Flores et al. 2010). It is permitted as food packing material in paper and wooden industries, mining ores, paints, and polishes. Additional major applications include oil recovery; gelatinization of xanthan in pumping operations, etc. (Kandra et al. 2018). Industrial production of xanthan gum is performed by spp. of Xanthomonas such as X. arboricola, X. axonopodis, X. campestris, X. citri, X. fragaria, X. gummisudans, X. juglandis, X. phaseoli, X. Vasculorium, and X. campestris (Petri 2015). Xanthomonas is a genus of Gram-negative tiny rod-shaped aerobes coming under the family Pseudomonadaceae. Many members of this genus are producers of xanthan gum, but most of them are phytopathogens (Vendruscolo et al. 2000). At commercial level, 70% of the substrate can be converted into the gum through aerobic fermentation at a temperature between 27 and 30  C, by using organic acids as stimulants. (Lopes et al. 2015). As a general practice, commercially exopolysaccharides are produced by batch fermentation and precipitation and centrifugation are the main techniques used for recovery of exopolysaccharides from the fermentation broth. Similar process is also applied in the xanthan gum production process. Studies have shown that the parameters and variables like composition of nutrients, techniques of feeding, temperature, pH, agitation, and foaming reduction can affect conversion of a raw material in xanthan (Rosalam et al. 2008).

4.1.2

Properties

Xanthan gum has the following important properties:

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1. High solubility: It has high solubility in both cold and hot water but barely soluble in maximum organic solvents. Highest solubility of xanthan gum is in 25% phosphoric acid and then in acetic acid, sulfuric acid, nitric acid, and sodium hydroxide. This propertiy varies according to the end use of the product; for instance, the bacteriological loads and heavy metal content for food application have to be very low although this is not the case for applications such as those in other industrial applications. 2. Highly viscous: It is very viscous even at low concentrations; even at a concentration of 1% it seems almost gel, yet pours readily and easy to mix and pumping. The viscosity is determined by temperature, the biopolymer concentration, amount of salts, and pH. 3. Emulsifying nature: It has notably high emulsifying, stabilizing, and suspending abilities. Xanthan gum exhibits great compatibility with thickeners present in the market such as cellulose derivatives, starch, pectin, gelatin, dextrin, alginate, and carrageenan. By combining various gums with xanthan gum in altered proportions. It performs better after modification with enzymatic treatments whch results in the removal of galactose residues (Kuppuswami 2014). 4. Low calorie: Similarly like other gums, xanthan gum is non-digestible in human gut and thus provides lower calories and easily passes through the gastric tract. 5. Pseudoplasticity: In solution, it has pseudoplastic nature; that is, the viscosity inversely changes with shear rate of a xanthan solution. This property enhances sensory potentials (flavor release, mouth feel) in food products and promises a high level of mixing, pumping, and pouring properties (Sharma et al. 2014). 6. High resistance to pH: It has a high tolerance to deviations in the pH range of 2–12 which makes it suitable to foods. Exceptional constancy is seen at low pH over long episodes of time. This property is vital for cleaners as well as for acidic food presentations such as salad dressings and fruit systems. 7. Thermostability: High resistance to temperature changes, even in the presence of acids and salts. It has brilliant freeze–thaw ability, viscosity which has stability up to 100  C, and even after heat treatment processes of sterilization, viscosity is maintained after chilling. It is evident that the pyruvate content of xanthan gum affects its thermal stability. Structure: The primary structure of xanthan gum, shown in Fig. 4.1, has a linear (1–4)-linked-D-glucose backbone with a trisaccharide side chain on every other glucose at C-3, containing a glucuronic acid residue linked (1–4) to a terminal mannose unit and (1–2) to a second mannose that connects to the backbone (Sanderson 1982). There is a repeated disachharide making backbone with right handed helix to form fiber. This conformation gives stability to the structure as the trisaccharide side chains are lined up with the backbone strengthened by non-covalent and hydrogen bonds. In solution, these side chains cover the backbone all over; in this manner, the (1–4) bonds are protected from attacks. It is believed that this shield is key to the excellent stability of the gum under hostile situations. Xanthan gum undergoes a conformational change on heating which is related with

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Fig. 4.1 Primary structure of xanthan gum

the conversion from an inflexible systematic state at low temperature to a more bendable and disordered state as the temperature increases (Pelletier et al. 2001).

4.2

Microbial Production of Xanthan Gum

The major operations involved in the microbial production of xanthan are as follows: 1. Organism and inoculum preparation 2. Media preparation 3. Fermentation

4.2.1

Organism and Inoculum Preparation

Industrial production of xanthan gum is performed by spp. of Xanthomonas such as X. arboricola, X. axonopodis, X. campestris, X. citri, X. fragaria, X. gummisudans, X. juglandis, X. phaseoli, X. Vasculorium, and X. campestris (Petri 2015) as shown in Fig. 4.2.

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Fig. 4.2 Generalized schematic for industrial production of xanthan gum

4.2.2

Media Preparation

Mostly naturally found raw materials are utilized for industrial production of xanthan gum. Media is diluted with tap water and carbon source generally used are glucose, sucrose, or starch and sometimes acid whey, up to a concentration of 5% as greater concentrations could suppress both growth and final production. To reduce the cost of media, its components are priorly sterilized. It is also beneficial to simplify the procedure, increase flexibility as modern process and equipment are

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used. Suspended constituents, such as soy protein, are not sterilized simply and thus cause fouling of the heated surfaces.

4.2.3

Fermentation

The production of xanthan gum starts with the fermentation consuming any suitable strain of Xanthomonas bacteria and substrate with sugar as carbon source, nitrogen, and salts. The fermentation broth results in containing 2–3% xanthan gum, which is further concentrated and purified up to 10% by UF. Thus, generated UF permeate stream if further treated by RO gives a pure water stream which can be recycled into the fermenter. Also, a concentrate containing xanthan gum, salts, and sugars is obtaained. The concentrated gum is then further refined by precipitating in methanol and the crude gum is recovered by decanter. Drying and milling is done to make a white free-flowing powder (Murad et al. 2019).

4.3

Factors Affecting Xanthan Gum Production

4.3.1

Effect of pH

It is assumed that optimum pH for XG growth is neutral. During the production of XG, pH decreases to 5 due to the presence of acid groups. Investigations reveal that after a fixed time duration pH of the broth keeps increasing. The pH value increased from 7 to 8 after 24 h and 8–9.5 after 48 h duration depends on the combination of temperature and agitation conditions. Conclusions say that pH conditions affect the growth of Xanthomonas not the production of xanthan (Psomas et al. 2007).

4.3.2

Effect of Temperature

It has been broadly studied how temperature affects production of xanthan gum. Gumus and researchers studied that the optimum temperature for XG production is 25–34  C while culture conditions require 28–30  C (Gumus et al. 2010). It was studied by Cadmus and researchers if temperature is increased for culture production, pyruvate content decreases. It is clear from the studies of Shu et al. that for the production of XG medium play as main pivotal factor for optimal temperature (Shu and Yang 1990).

4.3.3

Effect of Pressure

The pseudoplastic behavior and intrinsic viscosity of XG are reduced due to microfluidization. The effect of dynamic high pressure was studied by Laneuville and coworkers on XG in which they found that gel aggregates convert into solution

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with high shear force if applied once. When applied again, double-helical structure of XG was degraded into single chain. If the applied pressure is removed, XG will gain its original structure (Funahashi et al. 1987).

4.3.4

Effect of Carbon Sources

Carbon is the necessary component of culture medium. Every cell needs optimum concentration of nutrients and growth factors. Variation into nutrient and substrate concentration affects the side chain structure, molecular mass, and yield and not the backbone. Glucose and sucrose are the most common carbon sources in any medium. The carbon concentration for XG production is preffered to be 2–4%; concentration more than it prevents growth (Funahashi et al. 1987). Deacylation effect on rheological properties of xanthan–guar interaction is investigated by Khouryieh et al. and revealed that deacylated xanthan–guar gum mixture behave like gel whereas it is liquid like if the mixture is native. The reason behind that is destabilization of double-helical structure increased flexibility of side chain. Moreover, the deacylated XG contains more intrinsic viscosity than native. (De Vuyst et al. 1987).

4.3.5

Effect of Polymer Concentration and Salts

The XG solutions are more viscous if the concentration of XG is higher in the solution. The viscosity is produced due to its intermolecular bonding and reticulated structure. It has come into observation that the viscosity of XG solution may be changed by salt. Addition of small amount of salt lowers the viscosity of low concentrated solution of XG. This happens due to reduced electrostatic forces which otherwise causes an increase in viscosity resulting into less availability of molecules (Smith and Page 1982). Addition of salt into solution of high XG concentration causes more interaction of XG and salt molecules which result in higher viscosity (Palaniraj and Jayaraman 2011; Garcia-Ochoa et al. 2000).

4.3.6

Effect of Viscosity on Xanthan Gum in the Presence of Galactomannan

XG and galactomannan interaction causes an increase in viscosity of XG solution and also affected majorly by temperature. The dissolving temperature of galactomannan affects the viscosity and mesh size of the gel (Garcıa-Ochoa et al. 2000). It determines the maximum size of any passing molecule which affects the diffusion. Measurement of diffusion rate helps in its utilization for estimating the release kinetics.

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Applications of Xanthan Gum

Xanthan gum is reported to have enormous applications in many industries because it has some extraordinary properties such as longer stability of emulsions in acidic, alkaline, and saline mediums; thermal stability and pseudoplasticity (Fig. 4.3, Table 4.1). It has wide use in chemical industries such as xanthan and locust bean gum are used for manufacturing of deodorant gels. Due to high dispersion stability and low viscosity it is used to give a proper uniformity to the toothpaste (Palaniraj and Jayaraman 2011). By adding a minimum quantity of bivalent salts, xanthan gum can improve the color depth (K/S value) and acuity of the printed fabrics (Cao et al. 2019).

4.4.1

Pharmaceutical Applications

Xanthan gum is a stabilizer for suspensions of many unsolvable ingredients like barium sulfate (X-ray diagnoses), complexed dextromethorphan (for cough preparations) and thiabendazole. It is able to maintain homogeneity of liquids (Palaniraj and Jayaraman 2011), and it is reported to provide sustained release of

Fig. 4.3 Applications of xanthan gum in various industries

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Table 4.1 Summarizing different applications of xanthan gum Food application Salad dressing Bakery Beverage Preparation of food Soups, sausage, and gravies Dairy products Meat products Personal care Toothpaste Creams and lotion Shampoo Industrial application Agricultural products Cleansers Polishes Aqueous paints Textile and flooring print Glues Paper firms Ceramic glases Drilling of oil Biomedical Tablets

Usage in % 0.1–0.05 0.05–0.3 0.05–0.2 0.1–0.3 0.05–0.5 0.05–0.2 0.5–0.2 0.7–0.1 0.2–0.5 0.2–0.5 0.1–0.3 0.2–0.7 0.2–0.7 0.1–0.3 0.2–0.5 0.1–0.3 0.1–0.2 0.3–0.5 0.1–0.4

0.1–0.5 0.1–3.0

Function Easy pouring and good cling Improves texture Enhances taste Mixes fruit pulp Stabilizer Makes it tempting. Stabilize Stabilizer and emulsifier Holds water Easy pumping and good texture Stabilizer Improves solubility Proper mixing Provide stable pH range Mix abrasive components Control liquidity Improved color Control rheology and penetration Suspends particles Suspends solid Stabilizer Function recovery agent Provide excellent stability and good flow Retards drug release

References Sharma et al. (2006) Rosalam and England (2006)

Rosalam and England (2006)

Flickinger and Draw (1999) Katzbauer (1998)

Bindu and Ashok (2012)

drugs in the human system (Patel and Patel 2007), for example, XG-based pills of drug aceclofenac has been made by Ramasamy and colleagues for targeted drug delivery in colon. These tablets were stable at acidic pH of stomach and were degraded and absorbed in colon only (Ramasamy et al. 2011). Scientists have also formulated gastroretentive tablets using different hydrocolloids including XG using direct techniques with controlled physicochemical properties. These preparations confirmed to have sustained release for more than 20 h and stayed resilient for long. Similarly, Butani and colleagues formulated venlafaxine multifaceted matrix tablets using XG with rate controlling agent. Hence, it has been demonstrated by many authors that bursting of the tablets can be controlled by adding XG (Butani 2013). Scientists have established an antacid and antiulcer suspension using herbal extractions of Glycyrrhiza glabra, Terminalia chebula, Terminalia bellirica, Emblica officinalis, etc. and stabilized with various quantities of XG (Roopa et al. 2015).

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Food Industries

Xanthan gum is mainly applied as suspending and thickening agent in food industries of fruit juices and chocolate. It has already been approved by the US food and drug administration (FDA) for its use in food products. Most of the food items, nowadays need improvement of texture, look, flavor enhancement, viscidness, and liquidity. Xanthan gum is able to improve all these properties and additionally maintains the rheological performance of the foodstuffs. Also, it is useful to maintain pseudoplasticity and tempers the “gummy” taste than other gums (Palaniraj and Jayaraman 2011).

4.4.3

Dairy

When mixed with other polysaccharides such as galactomannans, carrageenan and guar gum are finer stabilizing agents for a lot of industrialized dairy foodstuffs such as milkshakes, ice milk, and ice cream. It has been used in manufacturing of frozen dairy products such as dessert puddings, acidified milk gels by mixing methylcarboxy methyl cellulose and CMC. These dairy stuffs are popular for being more stable, gentle, fresh in taste, viscidness, and tempting flavor (Flores-Candia and Deckwer 1999; Rosalam et al. 2008). With some other gums it is crucial for firmness, texture, and flavor of creamy cheese (Frank and Somkuti 1979) and karish cheese (Ahmed et al. 2005).

4.4.4

Bakery Products

Xanthan gum has been utilized for manufacturing bakery products as it lifts up the water holding capability throughout baking process and storage. It is also being used to increase shelf life of baked foods, as a replacement of eggs in some of bakery products, improves softness, air absorption, and holding in bread blends, biscuits, and cakes. The foods supplemented with xanthan gum are known to lessen calorie and gluten content in breads and increases shelf permanency, freezing and thawing stability in creams and fruit fillings (Murad et al. 2019). Due to its capability of producing highly stable emulsions, it is functional to make oil and non-oil-based sauces and ketchups (Koocheki et al. 2009). As it has stability to withstand in acidic, alkaline, and salty mediums and also possess thermostability, it is used to make products for a long shelf life. Besides having textural properties, the capability to maintain flavor for a longer period and defrosting constancy of xanthan gum has been exploited to derive very successful products (Bylaite et al. 2005). It is also applied in dairy goods due to stabilizing properties and protective effect against heat shock and prevent formation of icy crystals (Hemar et al. 2001). There are reports that adding xanthan gum was good to improve the firmness of cassava flour, taste, hardness, and crumbliness of the biscuits (Lu et al. 2019).

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Monthe et al. (2019) have demonstrated xanthan gum as a gluten substitute to make GF bread with a mixed flour prepared from fermented cassava, sorghum, and sweet potato and assessed their properties under varying parameters. Mohammadi et al. (2014) mixed xanthan gum and CMC to the blend of GF bread and found improved texture and taste. Kaur et al. (2015) reported that biscuits made from buckwheat flour with gums have more moisture, weight, size, and chunkiness and low breakage strength in comaprison to regular biscuits. The xanthan gum could considerably improve the look, color, flavor, and complete appropriateness of biscuits.

4.4.5

Beverages

In the processing of beverages, xanthan gum has been used as a bodying and dangling agent in drinks. It gives decent look and touch along with improvement in mouth feel to have pleasant taste, fast solubility, and compatibility with most components. In reconstituted beverage, it is used as taste enhancer which improves viscosity also (Murad et al. 2019).

4.4.6

Biomedical Application

Latest research shows that the xanthan gum is mixed with glycerides for improvement of performance of drilling fluids (Nunes et al. 2014). It is also mixed with magnetic nanoparticles and used as enhancer for the adsorption and removal of pollutant dyes from water (Mittal et al. 2014). It has appeared as an important tool in the biomedical field such as the intra-articular injection of xanthan are applied for treatment of osteoarthritis as it improves elasticity and provides lubrication between the joints (Han et al. 2012). Its high molecular weight is useful to build up physical and chemical networks, which is further used as carriers for drugs delivery and as scaffolds in biomedical tissue engineering. Due to acidic tolerance, it is used with some more polymers and mixed as excipient in tablets or as supportive hydrogels for drug release (Petri 2015).

4.4.7

Nanoparticle

In human lung cancer treatment, xatham gum is used to synthesize gold nanoparticle and these EPSs perform good to transport doxorubicin hydrochloride. (Pooja et al. 2014). Silver nanoparticles with increased catalytic and antiseptic properties for Escherichia coli and Staphylococcus aureus have been prepared with xanthan gum. (Xu et al. 2014). With other metals EPSs have also been used to synthesize nanoparticles because xathan gum reduces and stabilizes the reaction to produce palladium nanoparticles (Pd-NPs) with better catalytic activity through out the reaction of 4-nitrophenol to 4-aminophenol by sodium borohydride (Santoshi et al. 2015. For remediation of polychlorinated biphenyl- (PCB-) from contaminated soil,

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xantham gum is used with surfactant BriJ35 to stabilize iron, palladium NPs. (Fan et al. 2013). In food and non-food industries, xathan gum is commonly used for an increase in thickness and viscosity due to its softness. Xatham gum is also used as stabilizers in emulsion,suspensions, and foarms (Michel et al. 2003). It is also used with chitosan membranes in treatment of dermo-epidermal wounds (Michel et al. 2003; Bellini and Caliari 2014) and many pharmaceuticals (Gardin and Pauss 2001; Jagdale and Pawar 2014; Kuo et al. 2014; Razavi et al. 2014) and cosmetic industries (Jianlon and Yi 1999; Jamshidian et al. 2014).

4.4.8

Drug Delivery

Xanthan gum has enormous role in healthcare and drug delivery systems (Fig. 4.4, Table 4.2). It can be explained in the following points: Liposomes: Liposomes are spherical vehicles of lipid bilayer designed artificially to deliver both hydrophobic and hydrophilic compounds as they can protect entrapped compounds from degradation and release it on the targeted sites. Lipophilic barriers around the entrapped compound help in protecting it from enzymes, free radicals, and digestive juices. Stability of liposomes can be improved

Fig. 4.4 Potential role of xanthan gum in healthcare systems

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Table 4.2 Application of xanthan gum in drugs Application Treatment of posttraumatic corneal abrasions Buccal drug delivery

Drug Netilmicin

Application An eye gel with the antibiotic netilmicin

References Faraldi et al. (2012)

Zolmitriptan

Xanthum gum-based bilayer mucoadhesive Modified xanthum gum for treating sialorrhea

Advanced drug delivery Brain drug delivery

Lamivudine

Drug delivery

Carbamazepine

Wound healing Dermal patches

Metalloprotenase9 Paracetamol

Carbamazepine nanoemulgel for brain Wound healing by matrix metalloprotenase-9 For treating dermitis

Nasal drug delivery

Loratadine

Shiledar et al. (2014) Laffleur and Michalek (2017) Harika et al. (2011) Samia et al. (2012) Reiss et al. (2010) Gorle et al. (2017) Saudagar and Badhe (2016)

Cosmetic cream

Caffeine

Treatment of sialorrhea

Ph-dependent in situ nasal gel of loratadine using XG as a polymer Skin gel

Parente et al. (2015)

by using suitable polymers such as chitosan (Akbarzadeh et al. 2013). A positive effect of liposomal formulation was observed for pulmonary delivery by the liposome coating with polyelectrolyte complex formed by complexation of xanthan gum and chitosan (Sandolo et al. 2007; Manca et al. 2011). Hydrogel: Hydrogels are hydrophilic polymeric network chains which help in imbibing huge amount of water just like a biological tissue. Porosity of a hydrogel determines the drug loading capacity and the succeeding drug release. Porous structure can be regulated by cross-linking agents and polymer concentrations (Bindu and Ashok 2012). Superporous hydrogels are water-loving polymers with cross-linkage networks which improves their puffiness and absorption properties. Permeation and capillary action are the main modes of quick water absorption in a macromolecular structure. Superporous hydrogels can be prepared by using xanthan gum, 2 hydroxyethylmethacrylate and acrylic acid (Santos et al. 2005; Gils et al. 2009). It is observed that xanthan gum does not form hydrogels very fast instead these are only shaped when the aqueous solutions are taken to a particular temperature and immediately cooled (Sharma et al. 2006; Iijima et al. 2007). Matrix Systems: Matrix systems are composed of polymeric compounds which swell in the presence of water or biological fluids and can be used in controlled release of active drug ingredients. In matrix system, drug powder is evenly distributed in polymeric material matrix and compressed to form a tablet. The enlargement property of these polymers is helpful in the fragmentation of tablets and improvement of the termination rate of drug. According to Verhoeven et al.

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(2006) high enlargement and erosion rate, quicker drug release, and high absorption rate are the qualities of xanthan gum-mediated drug delivery in sustained release matrices. In colon drug delivery also when xanthan gum-mixed tablets show higher drug retarding capability than other formulations (Tiwari and Kumar 2009; Jackson and Ofoefule 2011). Xanthan gum is used in gum-based sustained release tablets and provides compatibility and inertness (Shinde and Kanojiya 2014). According to Vendruscolo et al. (2005) xanthan gum has increased the ability to hold back the drug release in comparison with synthetic cellulose derivatives. Niosomes: Niosomes are vesicular type of drug carrier transport system and have similarity with structure of liposome, but they are better in stability and are cheap in comparison to liposome. These possess both hydrophilic and hydrophobic parts so that both hydrophobic and hydrophilic drugs may be conveyed to the targeted sites. These can be used in eye drugs where their function is to carry hemoglobin and peptides (Ashutosh et al. 2012). Niosomes prepared with xanthan gum have changed particle size and good spreadability in comparison to the formulation without xanthan gum and the niosomal preparation have pseudoplasticity. The use of xanthan gum as a gelling agent to form serratiopeptidase niosome gel is proved experimentally (Vishakha et al. 2012; Shinde and Kanojiya 2014). Nanoparticles: Nanoparticles perform adsorbtion or encapsulation of the drug molecule for its protection from various biological degradation process, thus are very useful in drug delivery. The small size of nanoparticles [1–100 nm] makes them easily absorbable in comparison to larger molecules. Surface characters and the particle size of nanoparticles can be efficiently changed (Mohanraj and Chen 2006). Covalent, ionic, and poly bonds are the main types of linkages in the formation of polysaccharide nanoparticles (Liu et al. 2008; Vishakha et al. 2012). Gold nanoparticles synthesized using xanthan gum are nontoxic and biocompatible and also used to improve drug loading, stabilization, and increment of cytotoxicity in lung cancer cells (Wang et al. 2002; Pooja et al. 2014). According to a study, microand nanoscale iron particles can be stabilized by viscoelastic gel made of xanthan gum and guar gum mixtures (Williams et al. 1991; Xue and Sethi 2012). Microspheres: Microspheres are small spherical particles (1–1000 μm) used in the regulated delivery of unstable drugs. They can be applied in gene delivery, nasal drug delivery, oral drug delivery, and gastrointestinal drug delivery (Sahil et al. 2011). A 3D interpenetrating polymer network is developed by cross-linking of xanthan gum and polyvinyl alcohol and potentially delivered drug in a much sustained release pattern (Bhattacharya et al. 2013). According to a research, hydrophilic gums like xanthan gum and locust bean gum can be useful in delaying the drug release and enhance the duration of drug release (Deshmukh et al. 2009; Xue and Sethi 2012).

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Food Applications

XG is used as an additive in the food industry. It is biosynthetic and edible gum and at present additives are widely used for baking. Serum cholesterol is found to be lowered by the consumption of guar gum-like edible gums. In a study subjects of diabetes and without diabetes were chosen to assess the acceptability of XG (present in muffins) with a daily dose of 12 g in diet. Postload and fasting glucose level was found to be lowered after the XG consumption thus can be suggested that XG use into diet may help in the initial treatment of diabetes mellitus (Osilesi et al. 1985). In a study conducted by Preichardt et al., role of XG in maintaining the quality of gluten-free cakes was assessed for celiac patients. Three different concentrations of XG were taken for the formulations.Some authors analyzed the chemical, physical, and sensory features which revealed that XG combination improves quality of the characteristics such as enhanced and uniform features, delayed staling, and an increase in specific volume whereas both control and sample cake appeared same physically (Preichardt et al. 2011). In a study conducted for the examination of baked food quality having four different hydrocolloids, viz. Arabic gum, guar gum, methyl 2-hydroxyl ethyl cellulose, and XG. Evaluation of certain parameters such as degree of softening, dough development time, mixing tolerance index, and water absorbing capacity. Crumb hardness, baked food quality, and sensory evaluation were assessed. XG containing dough was found to be more water absorbing due to the presence of hydroxyl group in studies of water absorption capacity. In addition, loaves which contain guar gum were softer than that which were containing XG after 72 h of storage (Hui 2005). Another reason for the use of XG in food industry is its high viscosity even at a very low concentration which enables it to be used as a thickner. It shows pseudoplastic behavior in aqueous solution helping in pouring, filling, mixing and pumping, etc. (Geremia and Rinaudo 2005; Milas et al. 1990). Highly stable at variable pH, ionic strength, temperature and under shear when processed and packed (Hui 2005). For the prevention of water migration into pastry from the baked foods, xanthan gum is also used in bakery fillings. Recently, due to the elasticity of dough lent by XG, it is being added to gluten-free baked goods where its concentration range between approximately 0.05 and 0.7 wt % (Zhou et al. 2014).

4.4.10 Cosmetics In cosmetic industry also XG is used as an emulsion stabilizer, thickener, and texture enhancer. Due to the ability to make gel structure into water, XG is also used as viscosity modifier such as guar gum. XG is patented as fixative for the composition of cosmetics by Cao et al. In the study, it was revealed that heat treated XG containing composition performed better than the carbomer hair gel/polyvinyl pyrrolidone in wet and dry combs and stiffness, etc. (Cao et al. 2003). An eyeshadow containing XG mixed into dyestuff and silicate aqueous medium has been patented by Collin and his team. It can be stored at 45  C and remain homogenous for

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2 months due to the presence of gelling agent in the composition. Due to fluid-like texture, spreading on the eye lids as well as removal is easy and stays long (Collin et al. 2003). Formulation of bioadhesive using caffeine and XG as model drug and secondary polymer for skin application was carried out by Parente and coworkers which was characterized by adhesion, rheological, spreading capacity, and in vitro release studies. The revelation of in vitro studies was that release time for approximately two third of drug was very less. Other physicochemical studies were helpful in selecting optimum formulation (Parente et al. 2015). In a research by Saharudin along with coworkers examined what effect XG shows on some properties of rice bran oil such as physicochemical and rheological. By the use of emulsification process, six formulations were prepared. In studies, it was revealed that droplet size and zeta potential were increased with an increase in XG concentration. Author concluded that improvement in physicochemical properties of formulation can be done by using XG (Saharudin et al. 2016). Amnuaikit and his coworkers prepared a sunscreen lotion by using XG which was found to be having high sun protection factor (SPF) as well as both UV filters (organic and inorganic). In the formulation, XG was used in aqueous phase whereas UV filters were titanium dioxide and anisotriazine. In the evaluation results of prepared creams, in vitro SPF value was found to be more when the above-mentioned combination was used (Amnuaikit and Boonme 2013). Meanwhile at present, XG is present in various cosmetic products as well as toothpaste and some dermatological products (Singhvi et al. 2019).

4.4.11 Oil Industry Xanthan is also used in petroleum industry in extraction of oil (Katzbauer 1998) fracturing, pipeline cleaning, and work-over and completion. In thermal degradation, xanthan gum has good coordination with salt and tolerance that is why it is also used as an ingredient in drilling fluids (Khalil and Jan 2012). In micellar-polymer, xanthan gum is used as a tertiary oil recovery agent. Xantham gum is also used to fill the rock of sand in which oil is poured to make sure the proper extraction of oil. Xanthan gum is used in formulation of drilling fluid, starch modifications, and calcium products because of its high specific gravity. The drilling fluid’s thickness is managed by weighting the supplements which to make sure that hydrostatic pressure is same and fluid is not lost (Xia et al. 2020).

4.5

Conclusion and Future Prospective

XG is a natural gum which has an extraordinarily miscellaneous role in different branches of science. Particularly, it is capable of undertaking a range of chemical amendments with personalized properties. Because of such qualities, it is extensively used to make adhesives, gums, coatings, emulsions, and binders as it has been approved as a nontoxic gelling and stabilizing agent with wide industrial applications. It is a nonsensitizing compound which makes it suitable for use in

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drugs. On the ground of such properties, xanthan is approved by the FDA for use as a food stabilizer without any precise magnitude limitations and in pharmaceuticals with certain guidelines. It is shown that XG along with herbal powders can give better uniformity to tablets. It gives wide growth to industries; besides being natural, it has tolerance to generally practiced processing protocols and offers the advantages of biodegradability and low toxicity.

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Biosynthesis and Characterization of Poly-(3)-hydroxyalkanoic Acid by Bacillus megaterium SF4 Using Different Carbohydrates Temitope O. Fadipe, Nazia Jamil, and Adekunle K. Lawal

Abstract

The non-degradability of synthetic polymers of plastics has been associated with ecosystem imbalance. This requires the development of eco-friendly biopolymers. Poly-(3)-hydroxyalkanoic acids (PHAs) are microbially derived biopolymers produced by bacteria and have physical and chemical properties analogous to synthetic polymers thus making them candidates for bioplastic production. We share results from the use of isolate SF4 with potential for sustainable PHA production. The isolate was screened on PHA detection agar supplemented Nile red and Nile blue A and with Sudan Black B staining. It was biochemically characterized and identified by 16SrRNA sequencing. Production of PHA was done in a nitrogen-limiting medium containing 2% carbon source over 96 h. Extraction of PHA was by sodium hypochlorite/chloroform method and PHA was characterized by FT-IR and GC-MS. The PHA synthase genes, PhaC and PhaR, of the isolate were also partially amplified and sequenced. Orange and yellow fluorescence of PHA were observed for all carbon sources used. Blue-black intracellular inclusions of PHA were also observed after Sudan Black B staining. The isolate was identified as Bacillus megaterium SF4 sharing closest homology with B. megaterium NBRC15308 ¼ ATCC 14581. Growth T. O. Fadipe (*) Biotechnology Department, Federal Institute of Industrial Research, Lagos, Nigeria Department of Microbiology and Molecular Genetics, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan N. Jamil Department of Microbiology and Molecular Genetics, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan A. K. Lawal Biotechnology Department, Federal Institute of Industrial Research, Lagos, Nigeria # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_5

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curves showed highest biomass accumulation in 2% starch and PHA production of 26.53  0.91%. FT-IR spectra showed peaks corresponding to the presence of P3HB and P3HB3HV. Sequences of 16SrRNA, PhaC, and PhaR have been accessioned in the NCBI-GenBank as KY855376.1, KY855378.1, and MF947449.1, respectively. The results show that B. megaterium SF4 has great capacity for PHA production in varied carbohydrates. Keywords

Poly-(3)-hydroxyalkanoic acid · Biopolymers · Bioplastics · Bacillus megaterium SF4 · Sustainable

5.1

Introduction

5.1.1

Plastics

Plastics derived from petrochemicals have found broad applications in day-to-day human activities and in the industries because of their favourable mechanical and thermal properties (Ojumu et al. 2004). Their durability, flexibility, lightness, and resistance to degradation makes them more desirable than glass and wood (Aarthi and Ramana 2011). They are made from synthetic polymers such as polypropylene, polyvinylchloride, and polyethylene (Pathak et al. 2014). The annual production of petroleum-based plastics was estimated at over 300 million tons with over 150 million tons of synthetic plastics and plastic-derived materials utilized globally on an annual basis. The production of synthetic plastics is expected to increase to an estimated amount of 810 million tons by 2050 (Muhammadi et al. 2015; Kumar et al. 2020). These plastics are recalcitrant and resistant to microbial degradation and are reported to accumulate in the environment at the rate of 28 million tons yearly (Muhammadi et al. 2015). Their accumulation and persistence in the environment as well as very slow degradation have been associated with serious environmental pollution of land and water bodies. It has been reported that up to ten million tons of synthetic plastics find their way into the oceans yearly causing detrimental effects to marine life forms and disruption of the oceanic environment (Kumar et al. 2020). In addition, several health problems such as cancer and nervous system-related diseases have been associated with toxic by-products of synthetic plastics (Pathak et al. 2014; Muhammadi et al. 2015). In order to reduce these negative environmental effects and overcome these problems, synthetic polymers can be replaced with biopolymers such as polylactic acid, polysaccharides polymers, aliphatic polyesters, and poly-(3)-hydroxyalkanoic acid which have similar mechanical, thermal, and chemical properties as synthetic polymers (Muhammadi et al. 2015; Kumar et al. 2020). These biopolymers are becoming increasingly popular because they are biodegradable, environmentally friendly, and have widespread applications in the industries. In addition, the global

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bioplastic market is expected to reach a production volume of 2.44 million tons by 2022 (Kumar et al. 2020)

5.1.2

Poly-(3)-hydroxyalkanoic Acid: Discovery, Structure, and Classification

Poly-(3)-hydroxyalkanoic acids (PHAs) are a group of polymers synthesized as intracellular carbon and energy reserves by archaea, several bacterial species (Gram positive and negative), fungi, and plants (McCool and Cannon 2001; Kung et al. 2007; Mahmoud 2012; Muhammadi et al. 2015; Kumar et al. 2020). They can be accumulated to levels as high as 90–97% of dry cell weight in these organisms (Muhammadi et al. 2015). They are biodegradable and biocompatible and can be sustainably produced (Kung et al. 2007; Alkotaini et al. 2015). When disposed in the natural environment, they can be degraded by indigenous microbes within 4–6 weeks (Ali and Jamil 2016). The production of the first PHA, poly-(3)hydroxybutyric acid (PHB), as intracellular inclusions in Bacillus megaterium was described by Maurice Lemoigne and co-workers in 1926 (Verlinden et al. 2007; Muhammadi et al. 2015; Kumar et al. 2020). PHAs have been extensively researched and commercialized by some companies in countries such as the USA (Procter & Gamble), Germany (Biomer Inc.), China (Lianyi Biotech), and Brazil (PHB Industrial Company) (Suchada 2010) though its production cost is higher compared with other synthetic polymers. As a result, cheap carbon substrates are being exploited for PHAs production to reduce overall bioplastics production cost by up to 60% (Du et al. 2012; Naheed and Jamil 2014). A typical PHA granule is coated on the surface with a layer of phospholipid and proteins called phasins. These proteins are the major compounds in the interface of the PHA molecule. The layer of phasin proteins stabilizes and prevents coalescence of PHA granules in the cytosol. The phasins influence the number and size of PHA granule (Verlinden et al. 2007; Kabilan et al. 2012). A PHA molecule consists of 600–35,000 R()-3-hydroxyalkanoic acid monomers ranging from 3 to more than 16 carbon atoms. These monomers could have extra side chains of saturated, unsaturated, straight, or branched compounds containing aromatic or aliphatic side groups (Tan et al. 2014; Muhammadi et al. 2015; Kumar et al. 2020). During the formation of the PHA polymer, the carboxyl group of one monomer forms an ester bond with the hydroxyl group of the adjourning monomer. There are over 150 types of hydroxyalkanoic acids of PHAs are known, and this number continues to increase with various research progress. The molecular weight of PHA ranges from 2  105 and 3  106 Da which depends on the number of constituent monomeric unit, bacterial strain used, and growth conditions (Muhammadi et al. 2015). Typically. PHAs are classified based on the number of carbon atoms in the side chain length of a PHA monomer (Kabilan et al. 2012). Short chain length PHA (SCL-PHA) contains 3–5 carbon in their side chain (Kumar et al. 2020). Poly-(3)-hydroxybutyric acid (PHB) and poly-(3)-hydroxyvaleric acid (PHV) belong to this class (Verlinden et al. 2007). Medium chain length PHA (MCL-PHA)

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contains 6–16 carbon atoms and long chain length PHA (LCL-PHA) contains more than 16 carbon atoms) in their side chains. These classes of PHA are accumulated by a wide variety of bacteria (Kung et al. 2007). In addition, some bacteria such as Aeromonas strains are accumulate copolymers of poly-(3)-hydroxybutyric and hexanoic acid (Muhammadi et al. 2015). Thus, PHAs have found broad range application properties because of the variations in their physical, chemical, and mechanical properties (Kabilan et al. 2012).

5.1.3

Biosynthesis of Poly-(3)-hydroxyalkanoic Acid

Over 90 microbial genera including 300 bacteria species, isolated from different ecosystems, that are capable of accumulating PHAs granules intracellularly have been studied (Aneesh et al. 2017; Kumar et al. 2020). In addition, some PHA-producing bacteria are constructed as genetic recombinants (Du et al. 2012). The production of PHAs from bacterial systems is preferred to production from other living systems including fungi and plants because of their high accumulation ability (Kumar et al. 2020). Desirable qualities in any PHA-synthesizing bacteria are high growth rate, ability to use inexpensive carbon substrates, and high accumulation capacity (Naheed and Jamil 2014). Some known factors that affect PHA composition and yield are bacterial strain type, substrate (type and concentration), and growth parameters used during production. Thus to produce PHA cost-effectively, the use of an efficient bacterial strain is critical and has been at the centre of several research efforts (Valappil et al. 2007; Babruwad et al. 2015). The properties of PHAs including elasticity, biodegradability, and renewability strongly depend on the pathway of biosynthesis, monomer composition, and chemical structure (Kumar et al. 2020). Broadly, bacteria are divided into two major groups based on culture conditions required for PHA synthesis (Muhammadi et al. 2015). The first group of bacteria requires an essential nutrient limitation such as phosphorous, nitrogen, sulphur, or magnesium in the presence of excess carbon for PHA synthesis. Examples of bacteria in this group are Ralstonia eutropha, Pseudomonas oleovorans, and Protomonas extorquens and members of the Bacillus genera. The second group of bacteria does not require a nutrient limiting condition for PHA synthesis. The PHA polymer is accumulated during their growth phase. Examples of bacteria in this category are Alcaligenes latus, a mutant strain of Azotobacter vinelandii and recombinant Escherichia coli (Muhammadi et al. 2015). The major pathway of PHA biosynthesis in bacteria is catalysed by three enzymes. In the first reaction, two acetyl coenzyme A (CoA) molecules are condensed into acetoacetyl-CoA by β-ketothiolase encoded by phaA gene. The second reaction involves the reduction of acetoacetyl-CoA to (R)-3hydroxybutyryl-CoA by the NADPH-dependent enzyme, acetoacetyl-CoA reductase/dehydrogenase encoded by phaB gene. In the last step, (R)-3-hydroxybutyrylCoA monomers are used as precursors for the polymerization of poly-(3)hydroxybutyric acid (PHB) by a PHA polymerase encoded by phaC gene in an

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esterification reaction. The genes involved in this major pathway are assembled into the phaCAB operon (Sheu et al. 2000; Muhammadi et al. 2015; Kumar et al. 2020). In addition, some can also synthesize MCL-PHA through de novo fatty acid biosynthesis and β-oxidation pathways. This has been reported in members of the Pseudomonas genera (Muhammadi et al. 2015; Kumar et al. 2020). Over 20 other PHA biosynthetic operons have been cloned and analyzed in bacteria till date (Sheu et al. 2000). Based on the type of PHA polymerase, bacteria are grouped into four classes, namely class I made by Ralstonia eutropha, class II by Pseudomonas species, class III by Allochromatium vinosum, and class IV by Bacillus species (Rehm and Steinbüchel 1999; Ali and Jamil 2016). Class IV PHA polymerases are encoded by phaC and phaR genes which are both important for polymerizing activity in vivo and in vitro (McCool and Cannon 2001; Nayak et al. 2013). Several bacterial species belonging to the Bacillus genera including strains of Bacillus megaterium are known to utilize varieties of carbon sources for growth and PHA production as reported by several authors. They are able to use pure carbon substrates such as glucose, lactose, fructose, sucrose, (Shamala et al. 2003; Krueger et al. 2012; Okwuobi and Ogunjobi 2013; Medjeber et al. 2015), pure glycerol, waste glycerol (from biodiesel production), castor oil, waste frying oil, whey (Cardozo et al. 2016), sugarcane molasses, corn steep liquor (Gouda et al. 2001), beet molasses (Medjeber et al. 2015), and pure and waste cassava starch (Krueger et al. 2012; Medjeber et al. 2015; Aneesh et al. 2016). This study investigates the potential of a novel Bacillus megaterium strain isolated from cassava dumpsite for PHA sustainable production.

5.2

Materials and Methods

5.2.1

Detection of PHA Production by Isolate SF4

Bacterial isolate SF4, previously isolated from sugar farm site soils by Salaam et al. (2016), was used in this study. Poly-(3)-hydroxyalkanoic acid (PHA) production by isolate SF4 was detected following the viable colony staining protocol in a nitrogenlimiting and carbon-rich medium (2% of each carbon source: glucose, sugarcane molasses, glycerol, and starch). Isolate SF4 was first refreshed on nutrient agar for 24 h and pure colonies were cultured on PHA detection agar (PDA) containing potassium dihydrogen phosphate—1.33 g, ammonium sulfate—0.2 g, citric acid— 0.17 g, magnesium sulfate heptahydrate—0.12 g, agar—1.5 g, 1 mL trace elements solution supplemented with 0.5 μg/mL of Nile red or Nile blue A stains in a final volume of 100 mL. The petri-plates were incubated at 37  C for 24 h. For fluorescence detection of PHA, 24 h petri-plates were observed under an ultraviolet transilluminator and documented (Spiekermann et al. 1999; Fadipe et al. 2019a). Further screening for detection of intracellular PHA inclusions was done by Sudan Black B staining as described by Lee and Choi (1999). This was done by preparing heat-fixed smears of the isolate grown on PDA followed by staining with 0.3%

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Sudan Black B solution. Microscope slides were observed for blue-black intracellular inclusions under a compound light microscope and photomicrographs were taken.

5.2.2

Characterization of Isolate SF4

Isolate SF4 was streaked on freshly prepared nutrient agar plates and incubated for 24 h after which its colonial morphology was documented. Gram staining was done using heat-fixed smear of the isolate and its cellular morphology was observed under compound light microscope. Biochemical profile of the isolate was determined using the analytical profile index kit (BioMerieux). Other tests including catalase, oxidase, and starch hydrolysis were also done. Genomic DNA was extracted from 16–18 h bacterial broth culture according to the protocol in the Exgene™ Cell SV kit (GeneAll). This was followed by PCR amplification of the 16SrRNA hypervariable region and sequencing at first Base (Singapore). Sequence homology search was done using the NCBI-nucleotide BLAST tool and alignment was done with ClustalW in MEGA7. Phylogenetic analysis was done using Tamura-Nei algorithm of the neighbour-joining phylogeny method considering 1000 bootstrap replications in MEGA 7 (Fadipe et al. 2019b).

5.2.3

Assessment of Poly-(3)-hydroxyalkanoic Acid Production

This was done following the method described by Fadipe et al. (2019a). Isolate SF4 was grown overnight in a 250 mL flask containing 50 mL of nutrient broth at 37  C and 120 rpm. Seed culture was harvested by centrifugation at 6000 rpm for 10 min and washed in PHA broth medium (PBM) containing in 100 mL, potassium dihydrogen phosphate—0.33 g, ammonium sulfate—0.2 g, citric acid—0.17 g, magnesium sulfate heptahydrate—0.12 g, and 1 mL trace elements solution. The bacteria pellet was dissolved in 5 mL of PBM and inoculated into a 500 mL conical flask containing 300 mL of PBM (optical density 0.05–0.1 at 600 nm) supplemented with 2% of each carbon source (glucose, sugarcane molasses, glycerol, and starch). The flasks were incubated for 96 h at 37  C with shaking at 120 rpm. Sample (50 mL) were drawn every 24 h for PHA extraction. Bacteria-PHA biomass were collected by centrifugation at 6500 rpm for 10 min, and the resulting pellet was lyophilized to estimate the dry cell weight (DCW). The growth curve of isolate SF4 over the 96 h incubation period was monitored by measuring the optical density of the media at 600 nm. Each experiment was set up in duplicates conducted under aseptic conditions. Descriptive statistics and Pearson correlation coefficient were done using Microsoft Excel 2016 and results are expressed as arithmetic mean  STD.

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Extraction of PHA

Poly-(3)-hydroxyalkanoic acid was extracted following modifications to the methods of Ali and Jamil (2016) and Berger et al. (1989). Freshly prepared 5.25% sodium hypochlorite (NaClO), pH 10 was added to the lyophilized bacterial-PHA biomass and vortexed vigorously to obtain an evenly dispersed solution followed by incubation at 37  C and 120 rpm for 40 min. Labelled conical flasks containing chloroform was prepared and the bacterial-PHA-NaClO mixture was added onto it followed by incubation at 37  C with shaking at 120 rpm for 1 23 h. Using a glass separating funnel, the resulting the mixture was separated to collect the less dense phase containing PHA and chloroform. This was followed by concentrating the PHA-chloroform mixture in a Heidolph 16-G1B diagonal condenser rotary evaporator and drying of the concentrated mixture in a fume cupboard at room temperature. The weight of extracted PHA was estimated and percentage PHA of the DCW was calculated.

5.2.5

Characterization of PHA

Extracted PHA was characterized by Fourier transform infrared spectroscopy (FT-IR) and gas chromatography with mass spectrometry (GC-MS). Ten milligrams (10 mg) of extracted PHA was treated with KBr and subjected to FT-IR in an Agilent Cary 660 FT-IR spectrophotometer. The spectral range of 650–4000 cm1 and resolution of 2 cm1 and 32–64 scans was averaged for PHA functional group detection. The FT-IR spectra were acquired according to the KBr disc protocol (Ali and Jamil 2016; Fadipe et al. 2019a, b). The method of Ali and Jamil (2014) was used to determine the chemical composition of extracted PHA by GC-MS. Chloroform (1 mL), methanol (0.85 mL), and concentrated H2SO4 (0.15 mL) were added to 8 mg of extracted PHA followed by heating at 100  C for 140 min. One milliliter (1 mL) of the methanolysed PHA sample was injected into a Shimadzu gas chromatograph-mass spectrometer QP2010 in split injection mode (70:30) with nitrogen as the carrier gas at a flow rate of 3 mL/ min. The temperature of the oven was set at 60  C for 2 min at the start, ramped at the rate of 5  C per minute to 260  C and held for 15 min. The injector temperature was set at 260  C and column flow rate was 0.57 cm/s. A GC-MS QP2010 with D1 mass spectrometry system with ion source temperature set at 200 C was used to determine the monomeric composition of methanolysed PHA sample.

5.2.6

Amplification of PHA Synthases C and R Genes in Isolate SF4

The PHA synthase genes, PhaC and PhaR of isolate SF4 was amplified by end-point polymerase chain reaction (PCR) and amplicons detected on 2% ethidium bromide stained agarose gel in 1 TAE buffer. For the PhaC gene, BmphaC015F—

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CGTGCAAGAGTGGGAAAAAT and BmphaC931R—TCGCAATATGATCAC GGCTA primer pair with expected amplicon size of 900 bp were used in a gradient PCR reaction (Nayak et al. 2013; Fadipe et al. 2019b). For the detection of PhaR gene, PhaR4F—AAACCGAATCTTACTGGGAAG and PhaR4bR—GAAGCTG CTCACCTTGAGTTTTAAT primer pair were used (Fadipe et al. 2019a, b). The PCR cocktail, at a final volume of 50 μL, contained 25 μL 2 Dream Taq Green PCR master mix (Thermo Scientific), 0.5 μL of 100 μM forward and reverse primers, 19 μL nuclease-free H2O, and 5 μL DNA template. The PCR gradient cycling conditions for the detection of for PhaC gene was 95  C for 5 min initial denaturation, 95  C for 60 s denaturation, 55.61, 56.32, 57.28, and 58.32  C for 60 s annealing, 72  C for 2 min extension, 72  C for 5 min final extension, and hold at 4  C for 40 cycles in a Supercycler Trinity (Kyratec). The PCR cycling conditions for the detection of PhaR gene was 95  C for 5 min initial denaturation, 95  C for 60 s denaturation, 61.47  C for 60 s annealing, 72  C for 2 min extension, 72  C for 5 min final extension, and hold at 4  C for 40 cycles in the thermal cycler listed above. The PCR products were purified using the method detailed in the Expin™ Combo GP kit (GeneAll). The 900 bp (PhaC) and 432 bp (PhaR) fragments were sequenced using the 3730xl DNA analyser (Applied Biosystems) at Macrogen (Korea). Sequence homology search was done using the NCBI-nucleotide BLAST tool and alignment was done with ClustalW in MEGA7. Phylogenetic relationships with other closely related species were inferred using the predicted amino acids of PhaC and PhaR according to the Poisson model with the neighbour-joining method in MEGA7 considering 1000 bootstrap replications (Fadipe et al. 2019b).

5.3

Results and Discussion

5.3.1

Detection of PHA Production by Isolate SF4

Poly-(3)-hydroxyalkanoic acid production was observed as orange, blue, and yellow fluorescence with Nile blue A and Nile red stains on PHA detection agar which signifies PHA accumulation (Fig. 5.1). Additionally, orange fluorescence is indicative of the accumulation small chain length PHA, specifically polyhydroxybutyrate (PHB) (Spiekermann et al. 1999; Fadipe et al. 2019a). Sudan Black B stained heatfixed smears of isolate SF4 showed the presence of blue-black intracellular inclusions (Fig. 5.2) for the four carbohydrates used in this study (Lee and Choi 1999). This further confirms PHA accumulation and capacity of isolate SF4 to utilize these carbohydrates for PHA production (Tan et al. 2014; Ali and Jamil 2016; Salaam et al. 2016).

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Fig. 5.1 Fluorescence detection of PHA in B. megaterium SF4 using sugarcane molasses as carbon source. (a)—Nile blue, (b)—Nile red

Fig. 5.2 Intracellular detection of PHA in B. megaterium SF4 using glycerol and sugarcane molasses as carbon sources. GLY (a)—glycerol, SM (b)—sugarcane molasses

5.3.2

Colonial, Morphological, Biochemical, and Molecular Characterization of Isolate SF4

Colonial characteristics of isolate SF4 on nutrient agar was observed after 24 h incubation. Isolate SF4 colonies are creamy, unpigmented, circular, motile, and non-slimy with convex elevation and entire edges (Medjeber et al. 2015; Yuli et al. 2017). It is a Gram positive, endospore forming, and rod-shaped bacteria with cells appearing in clusters, chains, and pairs (Medjeber et al. 2015; Yuli et al. 2017). It was positive for glucose, mannitol, and sucrose fermentation and negative for the fermentation of inositol, melibiose, amygdalin, arabinose, rhamnose, sorbitol, and mannose. Furthermore, it was positive for catalase, oxidase, gelatinase tests and negative for urease test. Isolate SF4 was positive citrate utilization and starch hydrolysis and negative for indole production and hydrogen sulfide production (Medjeber et al. 2015; Yuli et al. 2017). Sequence homology with 16SrRNA nucleotide sequences on NCBI-nucleotide BLAST revealed that isolate SF4 shared closest evolutionary relationship with Bacillus megaterium NBRC15308 ¼ ATCC 14581 at 99 % identity score; thus, it is Bacillus megaterium SF4. The 16SrRNA sequences have been submitted in the

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Fig. 5.3 Phylogenetic tree of B. megaterium SF4 16SrRNA nucleotide sequences

NCBI-GenBank with accession number KY855376.1. Figure 5.3 shows Bacillus megaterium SF4 clustering with B. megaterium NBRC15308 ¼ ATCC 14581 (CP035094.1) in the phylogenetic tree below. The colonial, morphological, and biochemical characteristics of the isolate SF4 agrees with previous report by Yuli et al. (2017) expected for B. megaterium. The first documented bacterial-PHA producer, a Bacillus megaterium strain was identified by Lemogine in 1926 (Muhammadi et al. 2015; Kumar et al. 2020). In addition, several strains of B. megaterium have been documented as PHA producers (Nayak et al. 2013; Tan et al. 2014; Medjeber et al. 2015). The tree was constructed using the neighbour-joining model with Pseudomonas aeruginosa (red) as an outgroup considering 1000 bootstrap replications in MEGA7.

5.3.3

Growth Dynamics of Isolate SF4 in Four Different Carbohydrates

Figure 5.4 shows the growth curve of B. megaterium SF4 in the four carbohydrates used. In summary, the results show that PHA production in B. megaterium SF4 is growth related because PHA accumulation begins with cell growth in the log phase in all carbohydrates used (Aneesh et al. 2017). B. megaterium SF4 achieved the best growth in 2% starch supplemented PHA broth medium (PBM). It grew exponentially and peaked at 26 h followed by another sharp peak at 30 h and a sharp decline at 32 h. Thereafter, it grew steadily over the 96-hour incubation period. This observation agrees with reports by Medjeber et al. (2015) where the best growth rate was achieved by B. megaterium L9 using 2% starch as carbon source. In 2% glucose supplemented PBM, its growth increased steadily till it peaked at 24 h then achieved another major peak at 48 h and continued to grow gradually over the

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Fig. 5.4 Growth curve of B. megaterium SF4 in four carbohydrates as carbon sources. GLU— Glucose, GLY—Glycerol, SM—Sugarcane molasses, STA—Starch

96-hour period. In 2% glycerol supplemented PBM, its growth peaked at 28 h and another major peak at 50 h followed by continued increase throughout the experiment. In 2% sugarcane molasses supplemented PBM, its growth increased exponentially and peaked 24 h then grew steadily and remained constant.

5.3.4

Assessment of Dry Cell Weight and PHA Production in Four Different Carbohydrates

Table 5.1 shows extracted PHA and dry cell weight (DCW) in g/L and Fig. 5.5 shows extracted PHA in % DCW. We report that PHA production (% DCW) shows a weak-positive correlation with biomass accumulation (DCW in g/L) in glucose. However, there is a strong negative correlation between PHA production (% DCW) and biomass accumulation (DCW in g/L) when glycerol, starch, and sugarcane molasses were used as carbon sources. Figure 5.5 shows the extracted PHA from lyophilized B. megaterium SF4 biomass expressed as a percentage of its dry cell weight. Bacillus megaterium SF4 produced highest PHA amount of 4.69  0.17% in 2% glucose supplemented PBM at 72 h. We report a lesser yield compared to B. megaterium L9 which produced 50% PHA using glucose as carbon source after 48 h incubation at 30  C (Medjeber et al. 2015). Similarly, another strain of B megaterium which produced 39.9% using glucose as sole carbon source (Gouda et al. 2001). In contrast, we report a better PHA yield of 0.28  0.03 g/l and greater dry cell weight (DCW) of 6.13  0.04 g/L at 48 h (Table 5.1) compared to the 0.0143 g/L PHA yield and 0.0203 g/L DCW reported by Okwuobi and Ogunjobi (2013) using a strain of B. megaterium and

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Table 5.1 Mean dry cell weight and extracted poly-(3)-hydroxyalkanoic acid by Bacillus megaterium SF4 Carbon source Glucose

Starch

Glycerol

Sugarcane molasses

Time (h) 24 48 72 96 24 48 72 96 24 48 72 96 24 48 72 96

Avg. DCW (g/L) 3.07  0.16 6.13  0.04 3.83  0.47 4.56  0.45 1.77  0.10 2.82  0.42 5.58  0.40 6.12  0.03 1.77  0.01 2.35  0.35 3.70  0.34 6.55  0.18 1.47  0.18 3.10  0.00 3.38  0.03 4.10  0.11

Avg. PHA (g/L) 0.09  0.01 0.28  0.03 0.18  0.03 0.17  0.01 0.47  0.04 0.33  0.04 0.24  0.06 0.50  0.06 0.33  0.01 0.31  0.04 0.45  0.07 0.34  0.06 0.15  0.01 0.24  0.03 0.27  0.01 0.20  0.03

Fig. 5.5 PHA extracted produced by B. megaterium SF4 using four carbon sources

glucose as sole carbon source. In their study, the production medium was incubated 37  C for 48 h. The lesser PHA yield from our work and Okwuobi and Ogunjobi (2013) may be due to partial digestion of the lyophilized bacterial biomass or degradation of PHA during extraction with sodium hypochlorite (NaClO). Nicolas et al. (2008) have suggested that NaClO can serious degradations of PHA and up to 50% decrease in yield. A pretreatment by heating or freezing before NaClO

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treatment has been shown to improve PHA yields by up to 98% (Nicolas et al. 2008). Another factor which may have contributed to lesser PHA yield may be the higher incubation temperature of 37  C used in our study compared to 30  C used by Gouda et al. (2001) and Medjeber et al. (2015). The possible effect of a lower incubation temperature on dry cell weight and PHA yield using B. megaterium SF4 are worthy of further investigations. We report PHA amount of 26.53  0.91% and 1.77  0.10 g/l DCW in starch. This is lower than the yield of 73.46% PHA and 3.07 g/L DCW reported for B. megaterium PHB29 in starch at 48 h, pH 7.2, and optimum incubation temperature of 34  C (Aneesh et al. 2016). In addition, Medjeber et al. (2015) reported a maximum yield of 39.9% PHA and 0.815 g/L DCW in 2% after 48 h of incubation at 30  C with B. megaterium L9. Though we reported a higher amount of DCW compared to Medjeber et al. (2015), these discrepancies in DCW and PHA yield may be due to the higher incubation temperature of 37  C used in our study. Aneesh et al. (2016) reported a decline in PHA yield and DCW when incubation temperature was increased to 37  C and 40  C with B. megaterium PHB29 in 2% soluble starch. Krueger et al. (2012) investigated the PHA production potential of four B. megaterium strains, namely LAMA073, LAMA095, LAMA262, and LAMA265 using hydrolysed cassava starch by-product at 20% (v/v) and 40% (v/v) at 48 h and 37  C. B. megaterium LAMA095 produced the highest DCW of 4.48  0.5 and 4.97  0.9 g/L which translated to 23.88% and 29.78% PHA yield in 20% (v/v) and 40% (v/v) cassava starch, respectively. Their findings show that an increase in the starch concentration led to an increased DCW and PHA yield. In contrast, we recovered more PHA from a lesser DCW compared to the PHA yields and DCW reported in their work. These differences in yield may be due to different PHA extraction methods used in these studies. The use of sugarcane molasses as low-cost carbon source for industrial PHA production is been investigated because it is known to contain vitamins and trace elements such as riboflavin thiamine, pyridoxine, and niacin which are important growth factors for microbes (Gouda et al. 2001). Bacillus megaterium SF4 produced the maximum amount of 10.22  0.32% PHA (1.47  0.18 g/L) at 24 h and DCW of 4.1  0.11 g/L DCW at 96 h in 2% sugarcane molasses. Gouda et al. 2001 reported a PHA yield of 46.5% in 2% sugarcane molasses using a strain of B. megaterium. In addition, B. megaterium L9 produced 30% PHA in 2% sugarcane molasses (Medjeber et al. 2015). These yields are significantly higher than that from our study and may also be because of the lower incubation temperatures of 30  C used for PHA production in these studies. Bacillus megaterium SF4 produced the maximum amount of 0.33  0.01 g/L PHA and 1.77  0.01 g/L DCW at 24 h. Furthermore, a maximum amount of 6.55  0.18 g/L DCW at 96 h in 2% glycerol. Studies by Cardozo et al. (2016) reported a biomass of 1.23  0.05 g/L and a PHA yield of 2.80 g/L after 48 h for a Bacillus megaterium strain in a pH 7.0 production medium incubated at 30  C. We report a higher biomass of 2.35  0.35 g/L but a lower PHA yield of 0.31  0.04 g/L at 48 h. The discrepancies observed may be due to the lower incubation temperature used by Cardozo et al. (2016), a slightly higher concentration of 5.25% NaClO used

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in our study compared to 5% used by Cardozo et al. (2016) which may have caused some PHA degradation during extraction and other differences in our method of extraction. In general, we observed an increase in the number of spores in Sudan Black B stained cells as incubation time increased beyond 48 h for B. megaterium SF4. This suggests sporulation as a mechanism of survival under the limiting nutrient growth conditions thus leading to lesser PHA yield. A similar observation was described as a limitation when using spore-forming Bacilli for PHA production (Wu et al. 2001). However, sporulation can be prevented by maintaining a low pH in the medium during PHA production or adding up 90 mM α-picolinic acid to the PHA production medium (Akaraonye et al. 2012).

5.3.5

FT-IR Spectra Analysis of Extracted PHA

Figure 5.6 shows the FT-IR spectra of extracted PHA produced by B. megaterium SF4 using glucose (a), sugarcane molasses (b), glycerol (c), and starch as carbon sources (d). The four FT-IR spectra validate the presence of poly(3-hydroxybutyric) acid (Krueger et al. 2012; Gumel et al. 2012; Getachew and Woldesenbet 2016) and co-polymer of poly(3-hydroxybutyric) acid and poly3-hydroxyvaleric acid (Kumalaningsih et al. 2011; Shamala et al. 2009). Furthermore, they also reveal conformational changes in amorphous and crystalline phases of small chain length and medium chain length-PHAs in the extracted PHA (Gumel et al. 2012). The spectra show peaks at 2958, 2925, 2923, and 2916 cm1 which indicate irregular CH2 lateral chains (Gumel et al. 2012). Absorption peaks at 2842 cm1, 2823 cm1 are symmetrical methyl groups formed because of conformational changes during PHA crystallization (Sharma and Bajaj 2015). Peaks observed at 1721, 1722, and 1723 cm1 are the PHA characteristic bands corresponding to carbonyl stretching of esters. This indicates the crystallinity and chain length of the PHA (Krueger et al. 2012; Babruwad et al. 2015). Absorption peaks at 1459, 1458, and 1464 cm1 reveal the presence of CH2 and intracellular bacterial amide (-CO-N) groups (Getachew and Woldesenbet 2016). Further peaks at 1380, 1378, and 1376 cm1 reveal the presence of terminal methyl groups (Kumalaningsih et al. 2011; Krueger et al. 2012). The peaks observed at 1277 and 1276 cm1 are asymmetric ether groups stretching vibrations (Krueger et al. 2012; Getachew and Woldesenbet 2016). The peaks seen at 1230 and 1229 cm1 indicate asymmetric -C-O- groups (Kumalaningsih et al. 2011; Krueger et al. 2012). Additional peaks as seen at 1181, 1150, 1136, 1133, 1101, 1068, and 1056 cm1 represent C¼O stretching vibrations in the amorphous phase (Krueger et al. 2012; Gumel et al. 2012). More visible peaks at 981, 980, 930, 912, 829, 828, 812, 807, 806, and 805 cm1 correspond to alkyl halides in the extracted PHA (Getachew and Woldesenbet 2016).

Fig. 5.6 FT-IR spectra of PHA produced by B. megaterium SF4 using four carbon sources. (a) glucose; (b) sugarcane molasses; (c) glycerol; (d) starch

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5.3.6

GC-MS Analysis of Extracted PHA

Figure 5.7 shows the GC-MS chromatogram of extracted PHA produced by B. megaterium SF4. The spectra shows the retention time at which compounds were eluted. The highest peak was observed at 4.25 min. The chromatogram showed the main monomers as methyl esters of poly(3-hydroxybutyric) acid for all carbohydrates used in this study. Similar observation was reported by Ali and Jamil (2014). Table 5.2 shows the chemical composition with percentage abundance and molecular weight of PHA produced by B. megaterium SF4 as revealed by GC-MS analysis. In general, the effect of retention time on molecular weight (MW) was also observed as lighter compounds eluted first. However, an exception was observed for nitrobenzene (123.11 g/mol) which eluted after 1-hexanol 2-ethyl (130.23 g/mol). The most abundant compound was cyclohexanone with a percentage of 81.8% and MW of 98.14 g/mol. The least common compound was the oxaspiro cyclic ketone compound (7,9-di-tert-butyl-1-oxaspiro[4,5]deca-6,9-diene-2,8-dione) with 0.41%

Fig. 5.7 GC-MS chromatogram of PHA produced by B. megaterium SF4 Table 5.2 Chemical composition of PHA produced by B. megaterium SF4 shown by GC-MS S/ n 1 2 3 4 5 6 7

Retention time 4.25 5.06 7.83 13.55 16.15 19.38 20.10

Compound Cyclohexanone 1-Hexanol 2-ethyl Nitrobenzene Phenol, 2,4-bis(1,1-dimethylethyl) Diethyl phthalate n-Hexadecanoic acid 7,9-Di-tert-butyl-1-oxaspiro[4,5]deca6,9-diene-2,8-dione

% Abundance 81.80 9.96 2.54 0.74 2.31 2.83 0.41

Molecular weight (g/mol) 98.14 130.23 123.11 206.32 222.24 256.43 276.40

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abundance and MW of 276.40 g/mol. Our results agree with findings of Okwuobi and Ogunjobi (2013)

5.3.7

Characterization of PHA Synthase Genes

The PHA synthases of class IV PHA producers are encoded by the PhaC and PhaR genes among members of the Bacillus genera and are equally important for polymerization activity during PHA biosynthesis (McCool and Cannon 2001). The amplification of the 900 bp and 481 bp fragments of the PhaC (Fig. 5.8a) and PhaR (Fig. 5.8b) genes in B. megaterium SF4 confirms that it is indeed a class IV PHA producer (McCool and Cannon 2001; Nayak et al. 2013). Nucleotide sequences of PhaC and PhaR genes shared closest homology with B. megaterium NBRC15308 ¼ ATCC 14581 at 99% identity. Figure 5.9 shown below depicts the phylogenetic tree generated using predicted amino acid of the PhaC protein for B. megaterium SF4. In the tree, B. megaterium SF4 clusters with B. megaterium NBRC15308 ¼ ATCC 14581, Bacillus sp. ME40, and other B. megaterium strains. Figure 5.10 shows the phylogenetic tree generated using predicted amino acid of the PhaR protein for B. megaterium SF4. Similarly, the tree shows B. megaterium SF4 clustering other B. megaterium strains and Bacillus sp. Root239 and Bacillus sp. Root147. The accession numbers of the partial coding sequences of PhaC and PhaR genes for B. megaterium SF4 in the NCBI database KY855378.1 and MF947449.1, respectively.

Fig. 5.8 Agarose gel of SF4 PhaC and PhaR genes of B. megaterium SF4. L—1 kb DNA ladder, (a) 1–4 Gradient annealing temperatures; (b) 1 & 2—Gradient annealing temperatures

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Fig. 5.9 Phylogenetic tree of B. megaterium SF4 PhaC predicted amino acids sequences. The tree was constructed using the neighbour-joining model with Cupriavidus necator (red) as an outgroup considering 1000 bootstrap replications in MEGA7

Fig. 5.10 Phylogenetic tree of B. megaterium SF4 PhaR predicted amino acids sequences. The tree was constructed using the neighbour-joining model with Bacillus thuringiensis (red) as an outgroup considering 1000 bootstrap replications in MEGA7

5.4

Conclusion

Bacillus megaterium SF4 utilized various carbon sources for poly-(3)hydroxyalkanoic acid production. This potential should be further developed in temperature and pH optimization experiments for industrial application.

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Acknowledgments The authors acknowledge the support provided by ICGEB Arturo Falaschi SMART Postdoctoral Fellowship, S/NGA 16-01, and the Department of Microbiology and Molecular Genetics, University of Punjab, Lahore, Pakistan, for research collaboration on this work.

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Mushroom Mycelia-Based Material: An Environmental Friendly Alternative to Synthetic Packaging Abhik Mojumdar, Himadri Tanaya Behera, and Lopamudra Ray

Abstract

In the circular economy, reduction of the vigorous usages of nonrenewable resources is becoming the leading scenario. Fungal mycelium is the vegetative part of fungus consisting of a number of filamentous fibers that extend out of the fungus and is considered to be natural, fast growing, safe, and renewable. The ability to form self-assembling bonds helps them to grow quickly on biological and agricultural wastes and produce miles of thin fibers which bind to the substrate to form a strong biodegradable material and can easily be shaped for the production of packaging materials, architecture, and various new designed objects. With the benefit of cost-effective raw materials and sustainable substitute to polystyrene like hazardous synthetic materials, this mycelia-based material is becoming the material of choice. This chapter reviews the present scenario of technology-based mushroom cultivation using wastes generated from the agricultural industries and also focuses on a variety of utilizations as an alternative replacement for synthetic polystyrene. Keywords

Mushroom · Fungal mycelium · Packaging · Architecture

A. Mojumdar · H. T. Behera School of Biotechnology, KIIT Deemed to be University, Bhubaneswar, Odisha, India L. Ray (*) School of Biotechnology, KIIT Deemed to be University, Bhubaneswar, Odisha, India School of Law, KIIT Deemed to be University, Bhubaneswar, Odisha, India e-mail: [email protected]; [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_6

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Introduction

According to a report by the United Nations, approximately 68% of the total population will live in metropolitan areas by the year 2050, ultimately increasing the construction demands and other domestic uses. However, the natural resources are becoming scarce, demanding a search of newer renewable sources with alternative use of the existing resources. Before the fourth industrial revolution, the sustainability packaging in the industry has never been so vital. More than 350 lakh tons of paperboard packaging and 1600 lakh tons of plastic packaging are annually produced for several uses making their way through the supply chain. The packaging objects are disposed and become a part of the waste stream and again recovered by landfill disposal, recycling, and incineration. Recently, biodesign practices incorporating living organisms like bacteria, algae, or fungi, as essential components in design that can be grown rather than manufacturing are being an important area of research (Ghazvinian et al. 2019). Mushrooms can be cultivated on agricultural or industrial wastes and are considered as a major source of proteins, amino acids, along with many therapeutic applications. In industries, these can also be used for the biopulping and biobleaching applications. Previously, mushrooms were only considered to be a food, but different other value-added products can be obtained from the wastes such as twig or seed husks containing fungal mycelium. Mycelium can act as a natural glue, able to latch onto whatever around it such as plant stems or cotton shells creating a super-compact web of threads (Miles and Chang 2004). Therefore, the development of a packaging material with the mushroom mycelia can be designed as a cost-effective alternative over traditional plastic and other regular foams. Petroleum or natural gas-derived polystyrene foams are used widely in the prominent packaging, which is not biodegradable or compostable. So the incorporation of mycelium-based materials into the packaging base can help in the reduction of polystyrene consumption leading to eco-friendly packaging with enhanced sustainability and performance (Ashok et al. 2016; Vilaplana et al. 2010; Farmer 2013). The mushroom mycelium or the roots of mushroom can be converted into different shapes by growing them in a mould, and they also rapidly grow into a very compact material. After achieving the desired density with its shape, the dehydration step is done to halt further growth. After the effective life as a packaging material, these can be discarded in the expanse behind the house which can easily be decomposed automatically within a few weeks (Jiang et al. 2017; Abhijith et al. 2018). On the other hand, the use and production of polystyrene results in emission of greenhouse gases and is an energy-consuming process which eventually leads to environmental pollution. When non-recyclable and non-biodegradable polystyrene are used as single-use products such as food containers and food takeout plates, they play as the main element of marine debris and urban litter (Abhijith et al. 2018; Bruscato et al. 2019). The use of polystyrene and resin-based polypropylene is widespread because of many features such as easy recycling processes. However, these are not environment compatible and the processes are not sustainable.

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This chapter reviews the present approaches of the technology and also focuses on the mushroom mycelium-based packaging materials and cushioning/padding applications as a promising substitute for the polystyrene foam.

6.1.1

Demand of Ecological Modernization in the Packaging Industry

Since 1950, approximately 91,500 lakh tons of plastics have been manufactured worldwide, out of which 30% are in use, only 9% have been reused, and 60% are in disposal area as the discarded product (Geyer et al. 2017). According to a report, 2,86,000 lakh metric tons of plastic polymers will be produced by 2050 (Geyer et al. 2017). Though the fragments of plastics are known to be weakened by sunlight exposure, plastic packaging is not biodegradable and the impact of millimeter-sized fragments on the environment is still not fully understood (Tudryn et al. 2018). The price of plastic flctuates with the cost of the nonrenwable petroleum resources as theses reused in plastic manufacturing industries for is feedstock. For the transport of plastic manufacturers to downstream wholesalers, approximately 0.49 million British thermal units of energy is consumed with the emission of 0.04 metric tons of carbon dioxide for each ton of plastic wastes (Containers and Good 2016). The type of recycling process and the rate of conversion may differ depending on the type of plastic (Hopewell et al. 2009). Rate of the recycling changes according to the plastic types. For example, plastic water bottles comprising polyethylene terephthalate (PET) can be recurrently recycled with a restoration rate of 19.5% by weight (Containers and Good 2016), whereas the least recycled plastics include polystyrene and its variants such as extruded polystyrene (XPS) and expanded polystyrene (EPS) with a recovery rate of just 0.9% by weight. Moreover, the transportation cost of the polystyrene’s polymer structure is also costly and difficult making polystyrene an unattractive material for primary and secondary recycling. The waste-to-energy (WTE) combustion approach is the favored disposal method for plastic waste as compared to any other recycling process. But whatever be the treatment options, like WTE incineration, recycling including the collection of plastic fractions and transport, they harm the human health and environment. It is also apparent that no such promising waste management solutions and also searching for the same is quite challenging (Rigamonti et al. 2014). Due to increasing demands for consumer needs, rapid urbanization with changing consumer life style the expectation of improving the plastic sustainability in anticipation of creating a circular economy seems to be desolated (De los Rios and Charnley 2017). Moreover, only 5% of the retrieved plastics can be held costing around $80 billion and losses of approximately $120 billion annually (MacArthur et al. 2016). Therefore, in response to the highly rigid plastic markets in the 1980s plastic companies used blending options like the introduction of starch-based polymers to the polyethylene blends and also remarketed as “renewably sourced” (Iles and Martin 2013). Still, to meet the highest demand for plastic-based packaging, newer alternatives with null environmental impact and lesser cost needs to be investigated.

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Background of Bio-composite Based on Mycelium

6.1.2.1 Mycelium-Based Bio-composite? The vegetative section of the fungi is known as the mycelium composed of a mass of long, branched filamentous hyphae acting as a growth agent. Mycelium can break down the organic waste to simpler bodies with the enzymes secreted by the hyphae. This process lets the hyphae grow on the waste or any substrate, ultimately setting up a furry or condensed sheet around the substrate used known as “fungal skin” (Appels et al. 2019). The complete degradation process of the substrates is called colonization and during colonization mycelium binds the substrate replacing partially with strong biomass of the fungus. Removal of the fungal skin from the substrate can help in getting pure mycelium (Appels et al. 2019). Drying of the mycelium can stop the fungal growth leading its hibernation, which means at optimum environmental conditions the mycelium can restart growing. Similarly, the heating process permanently stops the growth of mycelium by killing it. However, the end products of either of the two processes are mycelium-based composites, and these can be framed by using non-carbon-based frameworks. The fungal species used for inoculation, growing conditions, substrates, additives used, processing and foaming techniques define the properties of pure and composite mycelium. Various reports are there comparing the properties of mycelium-based bio-composites with the conventional plastic (Appels et al. 2019; Haneef et al. 2017; Attias et al. 2017; Yang et al. 2017; Jones et al. 2017) such as (1) when compared to plastic, mycelium-based composite shows relatively low density, (2) low compact strength than plastics used for the construction projects, (3) reduced tensile strength, etc.

6.2

Early Uses of Mushroom Packaging

Irrespective of the size of the business and their margins, various strategies could be adopted to lessen the hazardous effects on the environment, emphasizing their sustainability. The packaging is very much necessary for the marketing of a product and almost all of the used packaging materials are discarded as litter once the sale is done. A small amount of consideration on using limited material at the time of packaging might support when it reaches to the shipping part. Mycelia are a natural binder and can be blended with agricultural wastes like oat and corn husks for making an extremely durable material and could be an option for replacement of styrofoam and polystyrene in every practical application. Ecovative pioneers the idea of the use of mushroom-based foam with intact maintenance of the key products. Dell Company has replaced its plastic packaging with mushroom material and announced that the shipping of its products will be by using eco-friendly mycelia foam. Vigorous tests had been carried out by Dell for ensuring the safety of their products similar to the traditional foam. Cushioning by using the mushroom materials proved to be a super green solution, and they are very easily composted after the use (Vendries et al. 2020). After Dell, Ford Company has decided on the

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application of foam containing mushroom as a major element in their automotive parts such as dashboards, bumpers, and side doors. Nearly about 30 pounds of petroleum-based foams are being used in each car and now the company would prefer for replacement of those with environmentally friendly alternatives. Similarly, Sweden-based flat-pack maker company Ikea has decided to lessen the use of fossilfuel-based material and mushroom-based foams are being considered as better substitute to polystyrene for their foam packaging (Vendries et al. 2020).

6.3

Potential of Mycelium-Based Material as an Alternative to Synthetic Packaging Materials

Advertising of the products focusing on their environment-friendliness was impressive particularly to a few committed group of buyers, but the gradual increase in the knowledge of the hazardous human effect on the fragile ecosystem has built a few development in viewpoints. The buying option of these environment-friendly packaging materials is mostly approved by younger generations, inspiring many multinational companies and local businesses. Now buyers are much more attracted towards reducing the waste production, and in buying consumable stuffs. The environmentally labeled packaging is recently considered as an important benchmark for the preference that can be professed as rising awareness regarding the significance of righteous and environmental dimensions in choosing the product. Some mushroom-based foam products are depicted in Fig. 6.1. Companies dedicating for ecological and social positivity get a significant rise in the sale. Both consumable and non-consumable goods claiming sustainability on their packaging exhibited a moderate 2% increase in their sell while brands without any sustainability claims showed only 1% of the increase in sell for their brands. Mushroom aids an extra way of the income to the cultivars as it makes a path for the utilization of their agricultural waste. Instead of remaining through generations, these materials are easily broken down in the landfills and compost. The area of packaging is positively affected by the use of mycelium, eventually obsolescing the use of toxic and persistent oil-based materials. According to the regional availability of the local agricultural by-product materials, the growth substrates of the mushroom may vary making it perfect for manufacturing all over the world. Regional manufacturing units using locally available feedstock can be used to reduce the cost of transportation for raw materials (Jiang et al. 2017; Stamets 2005). The big players in the global economy now remark these new trends. Companies like Unilever and Coca Cola have directed their attention towards making sustainable packaging enhancing their brand’s core values. Presently, world consumers are looking for a replacement to the hazardous packaging materials. Sustainable packaging practices will lead the business a path to tap into the attitude of buyers.

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Fig. 6.1 Mushroom mycelia based material foam used for packaging

6.4

Production of Mycelium-Based Material for Packaging

Myco-bond, one type of packging material that is resistant to heat and fire, biodegradable, low-energy material and ability to absorb energy has been developed by Ecovative, a start-up at New York by using mushroom mycelia. Compared to traditional foam packing material, only 12.5% of the energy and 10% of the carbon dioxide are required for manufacturing Myco-bond (a packing material). This is produced, allowing the mycelium of the mushrooms to invade the organic material like any agricultural wastes to feed on it (Vendries et al. 2020). There is a series of repeated growth and rupturing, then again greater growth resulting in a mass of mycelia which function almost the same as the polystyrene does in dry conditions, but when exposed to favoring environmental conditions suitable for breakdown, it decomposes easily. Mushroom roots and any other agricultural by-products such as corn or oat husk, together are kept in varied sizes of trays. These combinations are left for a minimum of 5 days in a dark warehouse to fester and furl. After which a fireproof, waterresistant, and biodegradable new packaging material emerges. Figure 6.2 shows a mushroom-based foam manufacturing facility. This approach is reframing in many ways which are discussed below: 1. Mycelium possesses unique properties of growing miles of a thread-like root in just 1 day. 2. The organism can grow amazingly very fast and fit into any mould-like dense foam.

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Fig. 6.2 Mushroom based foam manufacturing facility

3. These packaging materials can be used for almost everything such as packing for laptops, a wide panel for insulation at homes, and the density of each packaging material can be controlled by ending the growth process. The source material needs to be disinfected to remove any contesting spores present before starting the process. Disinfection by application of organic cinnamon oil, thyme oil, lemongrass oil, and oregano oil can be used instead of energyexhaustive steam sterilization process. Packaging foam of the Ecovative is produced by placing the organic wastes such as wood fiber or cotton hulls in a mould and then inoculated with the mushroom spores. Mushroom spores utilize the carbohydrate from waste and obtain energy for growing into the shape of the mould/tray. After reaching sufficient growth, the formation of flowering spores is stopped by subjecting them to heat. Since the mycelium is never allowed to grow for a longer period to form a mushroom, no spores or allergens left in this process (Vendries et al. 2020).

6.5

Mycelium Production and its Environmental Impact

Basal growth media such as sterilized mineral salt medium (MSM) enriched with supplements including carbon, nitrogen, and inorganic compounds was used as the production medium and with a 10% of inoculums (v/v) with a 6 days of incubation at 28  C with shaking at 150 rpm. Mycelia can be retrieved by centrifuging at 2500 g for 10–15 min. After the repeated washing steps, the recovered mycelia are dried and weighed until an unchangeable weight at 80  C (Zhu et al. 2015). Effects of pH, auxiliary carbon, and nitrogen source, presence of various inorganic compounds,

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and organic compounds like MgSO4, CuSO4, Zn (CH3COO)2 K2HPO4, and KH2PO4 are important for the growth of microbial mycelia under submerged fermentation conditions. Fermentation temperature, time, speeds of rotation, and inoculums size are some parameters which also affect the fermentation process (Zhu et al. 2015). Custom shapes of the mycelia are developed by self-assembling bonds formed between the mycelia by growing on the locally sourced agro waste products. Five variables, i.e., (1) active hyphal density, (2) density of the hyphal tip, (3) internal concentration of the substrate, (4) inactive hyphal density, (5) and external substrate concentration are used for defining the model. Branching architecture and filamentous growth habits of the fungi help them in adapting for their growth in the soil. During this process of growth, different types of vesicles providing new cell membrane moves forward, aiding the extension of hyphal tips. This hyphal tip movement with the secretion of lytic enzymes helps the hyphae to penetrate the solid substrate used like chitin and up taking of nutrients in the solute form. Redistribution of internal metabolites along the fungal mycelium is achieved by the translocation process (Olsson 1995). Hyphae which are involved in the uptake of nutrients, branching, and translocation are known as active hyphae and the tips are denoted as the end of these hyphae. Hyphae no longer involved in the above processes are denoted as inactive hyphae. Whatever may be the environment, a combination of elements and few elements like carbon, hydrogen, nitrogen, sulfur, oxygen, phosphorous, and other metals are required for their growth (Boswell et al. 2003).

6.6

Application of Mushroom Bio-composites

Previously, mycelia were only used in the medical industry to produce small molecules like antibiotics/enzymes/organic acids for more than a century (Wösten 2019). In the 1980s, Japanese scientist, Shigeru Yamanka first investigated the gluing power of the mycelium which can be effectively applied in the paper industry and in manufacture of many building materials. (Girometta et al. 2019). Recently, this mycelium is not only used for the binding and packaging materials but now it is used by many industries like design, fashion, packaging, architectural design, etc. In building sectors also, mushroom bricks were also tested for building 40 feet tower. Like the rigid board insulation, this mushroom-based foam can be used as an insulation material, providing a tight envelope with thermal bridges (Jones et al. 2017). Without the use of any toxic fire retardants, this can be used as a class A fire rating (Abhijith et al. 2018). MycoBoard is an engineered mushroom-based bio-composite, having the potential to replace wood material. Greensulate, a sustainable insulant and fireproof made from water, perlite, flour, and mushroom spores. MycoWorks and MOGU Company produced durable, flexible, and sustainable and waterproof synthetic leather (Fig. 6.3a) cultivated using mycelium. Officina Corpus coil started to develop a project “Future of Plastics” focusing on the development of kitchenware and alternatives to single-use plastics (Fig. 6.3b).

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Fig. 6.3 Applications of mushroom mycelia-based composites in product designing and architecture

Various furniture was designed from lightweight mycelium composite, regardless of their durability and load-bearing capacity (Fig. 6.3c). Apart from their use in designing industries, architectural industries also started exploring the applications of mycelium-based composites. Mycelium-based wall and ceiling panels were developed by MOGU (Fig. 6.3d). Over the past years, mycelium is used in building a few architectural projects like Hy-Fi Tower (Fig. 6.3e), the Mycotecture alpha (Fig. 6.3f), MycoTree (Fig. 6.3g). David Benjamine designed the temporary structure of the Hy-Fi tower in the form of three 13-m tall intersecting cylinders built with 10,000 blocks of mycelium (Slavin 2016). This design for the tower was for the experimental purpose to test the durability, biodegradability, and resistance of the mycelium-based masonry unit and the tower remained for 3 months in summer. Phill Ross, the cofounder of MycoWorks designed a small-scale pavilion Mycotecture Alpha. This pavilion consists of 350 mycelium blocks with mycelium floor also (Karana et al. 2018). Recently for the 2017 Seoul Biennale of Architecture and Urbanism, Block Research Group of ETH Zurich in collaboration with Karlsruhe Institute of Technology designed MycoTree by a 3D graphic static method. Besides their use of mushroom-based composite in the masonry architecture, they are recently attempted as a substitute to concrete in a construction project. The Shell Mycellium domed building has been constructed in India for Kuchi Muziris Biennale 2016. Various investigations on the use of mushroom-based material in architecture are moving forward rapidly. The main aim is to find a way for the affordance, enhanced

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durability, and improved strength against environmental threats and also to augment the chemical and mechanical properties of mycelium-based composites.

6.7

Conclusion

The review explains that fungi can degrade maximum of the organic, inorganic objects. Composites, based upon mycelium are now getting more attention as a sustainable alternative to the synthetic packaging, much new-concept bio-inspired design and insulation panels, etc. The challenges in scaling the rapidly increasing mycelia composite production are still to be looked upon. Overall the same reproducibility, mechanical and thermodynamic parameters could not be expected by these mushroom-based foams when compared to synthetic materials or monocomponent natural materials. However, suitable material characterization is still inadequate. Composites made up of mycelium, of which the investigation has only currently initiated, possess immense possibilities in replacing the synthetic hazardous packaging and in many other industries.

References Abhijith R, Ashok A, Rejeesh C (2018) Sustainable packaging applications from mycelium to substitute polystyrene: a review. Mater Today Proc 5(1):2139–2145 Appels FV, Camere S, Montalti M, Karana E, Jansen KM, Dijksterhuis J, Krijgsheld P, Wösten HA (2019) Fabrication factors influencing mechanical, moisture-and water-related properties of mycelium-based composites. Mater Des 161:64–71 Ashok A, Rejeesh C, Renjith R (2016) Biodegradable polymers for sustainable packaging applications: a review. IJBB 1(11) Attias N, Danai O, Ezov N, Tarazi E, Grobman YJ (2017) Developing novel applications of mycelium based bio-composite materials for design and architecture. Proceedings of building with biobased materials: best practice and performance specification, 6–7 September, pp 76–77 Boswell GP, Jacobs H, Davidson FA, Gadd GM, Ritz K (2003) Growth and function of fungal mycelia in heterogeneous environments. Bull Math Biol 65(3):447 Bruscato C, Malvessi E, Brandalise RN, Camassola M (2019) High performance of macrofungi in the production of mycelium-based biofoams using sawdust—sustainable technology for waste reduction. J Clean Prod 234:225–232 Containers P, Good ND (2016) Documentation for greenhouse gas emission and energy factors used in the waste reduction model (WARM) De los Rios IC, Charnley FJ (2017) Skills and capabilities for a sustainable and circular economy: the changing role of design. J Clean Prod 160:109–122 Farmer N (2013) Trends in packaging of food, beverages and other fast-moving consumer goods (FMCG): markets, materials and technologies. Elsevier Geyer R, Jambeck JR, Law KL (2017) Production, use, and fate of all plastics ever made. Sci Adv 3 (7):e1700782 Ghazvinian A, Farrokhsiar P, Vieira F, Pecchia J, Gursoy B (2019) Mycelium-based bio-composites for architecture: assessing the effects of cultivation factors on compressive strength. In: The eCAADe and SIGraDi Conference, University of Porto, Portugal

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Girometta C, Picco AM, Baiguera RM, Dondi D, Babbini S, Cartabia M, Pellegrini M, Savino E (2019) Physico-mechanical and thermodynamic properties of mycelium-based biocomposites: a review. Sustainability 11(1):281 Haneef M, Ceseracciu L, Canale C, Bayer IS, Heredia-Guerrero JA, Athanassiou A (2017) Advanced materials from fungal mycelium: fabrication and tuning of physical properties. Sci Rep 7(1):1–11 Hopewell J, Dvorak R, Kosior E (2009) Plastics recycling: challenges and opportunities. Philos Trans R Soc B Biol Sci 364(1526):2115–2126 Iles A, Martin AN (2013) Expanding bioplastics production: sustainable business innovation in the chemical industry. J Clean Prod 45:38–49 Jiang L, Walczyk D, McIntyre G (2017) A new approach to manufacturing biocomposite sandwich structures: investigation of preform shell behavior. J Manuf Sci Eng 139(2) Jones M, Huynh T, Dekiwadia C, Daver F, John S (2017) Mycelium composites: a review of engineering characteristics and growth kinetics. J Bionanosci 11(4):241–257 Karana E, Blauwhoff D, Hultink E-J, Camere S (2018) When the material grows: a case study on designing (with) mycelium-based materials. Int J Des 12(2):119–136 MacArthur DE, Waughray D, Stuchtey M (2016) The new plastics economy, rethinking the future of plastics. In: World Economic Forum Miles PG, Chang S-T (2004) Mushrooms: cultivation, nutritional value, medicinal effect, and environmental impact. CRC Press, Boca Raton Olsson S (1995) Mycelial density profiles of fungi on heterogeneous media and their interpretation in terms of nutrient reallocation patterns. Mycol Res 99(2):143–153 Rigamonti L, Grosso M, Møller J, Sanchez VM, Magnani S, Christensen TH (2014) Environmental evaluation of plastic waste management scenarios. Resour Conserv Recycl 85:42–53 Slavin K (2016) Design as participation. J Design Sci Stamets P (2005) Mycelium running: how mushrooms can help save the world. Random House Digital, Inc., New York Tudryn GJ, Smith LC, Freitag J, Bucinell R, Schadler LS (2018) Processing and morphology impacts on mechanical properties of fungal based biopolymer composites. J Polym Environ 26 (4):1473–1483 Vendries J, Sauer B, Hawkins TR, Allaway D, Canepa P, Rivin J, Mistry M (2020) The significance of environmental attributes as indicators of the life cycle environmental impacts of packaging and food service ware. Environ Sci Technol 54(9):5356–5364 Vilaplana F, Strömberg E, Karlsson S (2010) Environmental and resource aspects of sustainable biocomposites. Polym Degrad Stab 95(11):2147–2161 Wösten HA (2019) Filamentous fungi for the production of enzymes, chemicals and materials. Curr Opin Biotechnol 59:65–70 Yang Z, Zhang F, Still B, White M, Amstislavski P (2017) Physical and mechanical properties of fungal mycelium-based biofoam. J Mater Civ Eng 29(7):04017030 Zhu W, Guo C, Luo F, Zhang C, Wang T, Wei Q (2015) Optimization of Calvatia gigantea mycelia production from distillery wastewater. J Inst Brew 121(1):78–86

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An Overview of Microbial Derived Polyhydroxybutyrate (PHB): Production and Characterization Monika Sharma and Harish Kumar Dhingra

Abstract

The glory of synthetic polymer and the production and application can be contributed to their prize, permanence, and endowment for the proficiency in daily living. Although, one time utilization of synthetic plastic, longevity and rebellious characteristics is opened a continuous enchancement in synthetic polymer as a part of garbage. Requirement for the replacement of one-time utilized plastics which is hard to detect has motivated many researchers to work regarding the permanent solution to replace synthetic polymers that are oil-based plastics. Poly-β-hydroxybutyrate is a naturally formed biopolymer that can be used as plastic produced by various microorganisms. It has aquired importance because of its structural variations and similar analogy to synthetic polymer. Additionally, specific physico-chemical, bio-logical, and degradation characterisitics of biodegradable plastics is key factor which make bioplastic important for various uses in different field. This chapter contains classification, biosynthesis, detection techniques, and recovery methods of microbially produced biodegradable polymer. We then discuss the application of biodegradable polymers in various fields to reduce the dependence on synthetic plastic. Keywords

Biodegradable polymers · poly-β-hydroxybutyrate · biodegradation · bioplastic

M. Sharma (*) · H. K. Dhingra School of Liberal Arts and Sciences, Mody University of Science and Technology, Lakshmangarh, Rajasthan, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_7

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7.1

Introduction

7.1.1

Plastics and Problems with Plastics

Macromolecules made up of small molecules present in repeating structure are known as polymers. Polymers are either natural or man-made compounds. Polymers may be classified as homopolymers and heteropolymers on the basis of monomer units. Plastic is oil derived, partially man-made and organic compound which utilized in different kind of product formation. Plastics have many properties like light in weight, strong, and durable. Plastic is also resistant to microbial and chemical decomposition which increases its valuablilty for mankind. Plastic was introduced in 1862 by Alexander Parkes (Lemoigne 1925). Plastic is an important part of our everyday life. Plastic is used to make simple carry bags and complicated medical and surgical implants. Modern lifestyle increases higher use of one-time-use plastics. It is also used for various storage substances like milk, H2O, coke, extracts, and fruits. It increases consumption of oil-based polymers for storage packets. An upscaled use of man-made polymer increases the resistant waste that is not able to decompose by microbial and chemical decomposition mechanism. Depolymerase enzyme is absent and high molecular weight is high. These characteristics make synthetic plastic sustainable for decomposition. Supervision of oil-based polymers enhances work for garbage managing agency. Waste management authorities either discharges garbage in the earth surce/ocean or burn that reduced the productivity of land/ ocean and cause the air pollution. Different kinds of harmful compounds like HCN produced as a result of burning being the reason for the decline in a person’s physical and mental well-being. These synthetic polymer-made packets are dangerous for cattles. The reuse of synthetic polymer is an extremely expensive activity. Very less synthetic polymer carriers get reused. A lot of aquatic creatures and many waves pass on due to synthetic polymer carrier (Heap 2009). The plastic litter ingested by animals clogs their digestive tract which leads to death due to starvation. Birds entangled in synthetic plastic bags cannot fly and as a result they die. In sight of many dangerous effects of synthetic plastics, there is an urgent need for biodegradable plastic, which does not lead to such problems. Bio-based and biodegradable polymers can added to better economical condition by utilization of sustainable assets. In addition, organic polymers give new finish of life as an-aerobic degradation or fertilizing the soil that have lower or no negative impact on the environment (Narancic et al. 2020). The bio-based plastics have the following advantages over oil-based plastics: • Bio-based plastics prevent land-filling and various environmental pollutions. • Bio-based plastics can increase the organic part of the soil; retain water and nutrients in the soil. • Production of most of the bio-based plastics requires lower energy compared to synthetic plastics. • It reduces recycling cost and environment cleaning from the contaminated products.

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• The renewable source of bio-based polymer will conserve the nonrenewable sources of fossil fuels for a more sustainable enviroment and thereby reducing the emission of greenhouse gas (Pikon and Czop 2014).

7.1.2

Biodegradable Polymers

Biopolymers are also known as natural polymers, and they are produced from the polymerization of monomers from different metabolic process. The biopolymers have been classified into eight classification as follows (Steinbuchel and Hein 2001): • • • • • • •

Nucleic acids Polyamides Polysaccharides Polyhydroxyalkanoic acids, polymalic acid, and cutin Inorganic polyesters with polyphosphate Poly isoprenoids like gutta-percha Poly phenols like lignin

The biodegradable polymers of different classes do not have same properties. Out of these classes, a few biopolymers can be used as plastic. The classes of biodegradable polymers like polythioesters and organic polyoxoesters can act as a source of biodegradable plastics.

7.1.3

Biodegradable Plastics

A biodegradable plastic should be degraded through photo-degradation, oxidation, and hydrolysis without discharging any harmful residue into the environment (Swift 1993). Biopolymers are decomposed by lysis without enzyme activity and hydrolysis with bacterial enzymes (Poirier et al. 1995a, b). Polylactic acids (PLA), polyhydroxyalkanoates (PHA), and poly-propylene are various types of bioplastics (Patwardhan and Srivastava 2004; Datta et al. 1995). Out of them polyhydroxyalkanoate is a highly sustainable bioplastic that will decompose completely.

7.2

Polyhydroxyalkanoates

A number of biopolymers are formed in nature such as nucleic acids, polyamides, polysaccharides, polyoxoesters, polythioesters, polyanhydrides, polyisoprenoids, polyphenols, and polyhydroxyalkanoates (Steinbuchel and Hein 2001). Among the most investigated biodegradable polymers, polyhydroxyalkanoates are promising to other biopolymers because of their close analogy to plastics and structural variations (Kalia et al. 2007). These polyesters are used in a number of applications and have

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attracted considerable industrial attention (Madison and Huisman 1999). Hence, these polymers are acquired as alternatives to synthetic plastics. A wide variety of Gram positive and Gram negative, aerobic, anaerobic and photosynthetic, lithotrophic, and organotrophic bacteria accumulate PHA intracellularly as carbon and energy source (Senior and Dawes 1973). PHAs are polyesters of various hydroxyalkanoates and hydroxylated at various positions such as 3, 4, 5, and 6. A big amount of different monomer units which can be incorporated in this branch of biopolymer (Anderson and Dawes 1990). Approximately 150 different hydroxyalkanoate monomers have been recognized with various types of out-group structures. All of these are (R)-form chiral molecules that are synthesized by a wide range of microorganisms (Lee 1996a, b; Doi et al. 1992).

7.2.1

Classes of Polyhydroxyalkanoates

Polyhydroxyalkanoates are identified as small chain length PHA, medium chain length PHA, and long chain length PHA based on the size of the hydroxyalkanoic acid units (Steinbuchel and Pieper 1992). The small chain length PHA is made up of three to five units of 3 hydroxy or 4 hydroxy fattyacids. Poly-β-hydroxybutyrate (PHB) is the first of the PHA identified. Mcl and lcl-PHA are composed of C6 to C14 and more than C14, 3-hydroxy fatty acids, respectively. General structural formula is shown in the figure given below (Fig. 7.1 and Table 7.1): An enzyme for the synthesis of poly-beta-hydroxyalkanoates A. eutrophus is able to accept 3-HA with 3–5 C atom rather than enzyme of P. oleovorans is able to polymerize 3-HA with 6 to 14 C atom (Khanna and Srivastava 2004). Fig. 7.1 Structure of polyhydroxyalkanoates

Table 7.1 Representative members of polyhydroxyalkanoates (Steinbuchel 1991)

N 1 1 1 1 2 2 3 3 4

R H2 CH3CH3CH2CH3CH2 CH2H2 CH3H2 CH3CH3(CH2)4CH2-

Types of monomers Poly 3-hydroxypropionate Poly 3-hydroxybutyrate Poly 3-hydroxyvalerate Poly 3-hydroxyhexanoate Poly 4-hydroxybutyrate Poly 4-hydroxyvalerate Poly 5-hydroxyvalerate Poly 5-hydroxyhexanoate Poly 6-hydroxydodecanoate

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Microbes can produce PHA with small chain length and medium chain length groups which contain 3 to 14 carbon atoms have been reported. Scl PHA has properties similar to synthetic plastics like poly-propylene, while mcl-PHA shows more elastomeric characteristics (Gopi et al. 2018). These copolymers have characteristics between small chain length and medium chain length PHA on the basis of molecular partition of small chain length and medium chain length units that enhance mechnical and thermal characteristics. Insertion with a small number of medium chain length units, 3 hydroxyhexanoate in biopolymer basic structure, modifies the characterisitics of poly-beta-hydroxyalkanoates The formed polybeta-hydroxyalkanoates is closest to lower molecular weight poly-ethylene because of this being used at industrial level (Philip et al. 2007; Madison and Huisman 1999; Wang et al. 2007).

7.3

Poly-b-hydroxybutyrate (PHB)

The first and most commonly discovered PHA was poly-β-hydroxybutyrate (PHB). The PHB is nontoxic, biocompatible, and biodegradable plastics that are made up of renewable resources (Reddy et al. 2003). PHB is glassy in nature, rotate the plan polarized light and unsolvable in polar solvants. Properties like this derive PHB very reasonably as a petroleum-based polymer (Lee et al. 2000; Park et al. 2002a, b). Lemoigne (1925) identified homopolymer poly-β-hydroxybutyrate (Jackson and Srienc 1994). Macrae and Wilkinson (1958) observed that PHB was accumulated as a result of imbalance growth produced by nutrient limitation. Senior and Dawes (1973) reviewed on storage polymers and emphasized the role of polyhydroxyalkanoates as carbon storage compounds in microorganisms during nutrient depletion or limiting conditions. Poly-β-hydroxybutyrate synthesized by various Gram-positive and Gram-negative bacteria like Azotobacter sp., Rhizobium sp., Pseudomonas sp., and Bacillus megaterium, formed PHB molecules are stored as inclusion body inside the microbial cell (Steinbuchel and Valentin 1995). Plastics made up of PHB have been reported to be completely biodegradable. Microorganisms from various taxa like aerobic, anaerobic, photosynthetic microbes and archae bacteria can produce PHA (Luengo et al. 2003; Merugu et al. 2010). More than 300 species of bacteria are known to biosynthesize PHB (Reddy et al. 2003; Steinbuchel and Schlegel 1991; Suriyamongkol et al. 2007). Within the bacterial domain most of the PHB producers belong to the phylum proteobacteria (Rehm 2007). PHB producers are also found among cyanobacteria (Synechococcus), firmicutes (Bacillus), etc. PHB-producing strains have been isolated from diverse environments like activated sludge (Byrom 1987; Takeda et al. 1995; Lee et al. 1995; Akar et al. 2006), marine waters (Sabirova et al. 2006; Shrivastav et al. 2010), hypersaline environments, oil-contaminated soils (Dalal et al. 2010; Saharan et al. 2014), rhizosphere (Madison and Huisman 1999; Mohapatra et al. 2015), etc.

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PHB- producing Bacteria

A. vinelandii was not suitable for production at industrial level because it has a low yield of PHB produced. A. vinelandii UWD was important because it transformate without outer layer and accumulate 80% PHB (Page 1992). Byrom (1992) worked on the commercial isolation of PHB in the UK. Microbes of interest were from the family Ralstoniaceae at a lab in the UK due to extraction of PHB was easy and had good physical properties. PHB by methylotrophs and Azotobacter was also studied. Although that PHB was low in molecular weight and yield was hard to isolate also. Azotobacter utilized organic source to produce sugar at the place of PHB; hence, it was not an organism of interest. Ralstonia eutropha formed approximately 80% PHB when phosphate is present in restricted amount. The important microbes are those that can produce PHB while cultured on cheap materials. Lee (1996a, b) discovered that the study of microbial growth pace and PHB production pace by this organism is important for choosing potent microorganism for PHB production. The production of copolymer by 13 microbes from the rRNA superfamily III is discovered (Renner et al. 1996). They suggested that various microbes could synthesize polymer which contains various PHA combinations when cultured on similar materials. Rawte and Mavinkurve (1998) identified PHA-forming microorganisms from mangroves of Mandovi estuarine. Bacteria were primarily identified at tributyrin medium to check PHB production with the help of Nile blue dye under microscope. From 65 PHA-accumulating strains, some of them could utilize enviroment N2 then develop nitrogen-less medium. Lokesh et al. (2005) isolated PHB-producing bacterial species and characterized higher PHB producer as Sphingomonas sp. The bacteria were developed with various carbon sources and amount of produced polymers were checked. This bacterium might produce PHB when feed on disaccharides, aldo hexose, and sugar alcohols, but failed to accumulate PHB when cultured with ketoses, pentoses, and starch. Aerobic, free-living, and nitrogenfixing microbes isolated by Pal and Paul (2000) were identified as genus Azotobacter; nearly 70% of strains could produce biopolymer. The sustainable strain of A. chroococcum has been shown to store the PHB approximately 70% dried biomass, concurrent cultured on enriched parameters. Mercan et al. (2002) studied production of PHB in different species of Rhizobium (Rhizobium cicer, Rhizobium japonicum, Rhizobium sp., and Bradyrhizobium japonicum). In the study, they found that Rhizobium sp. 2426 strain can produce PHB up to 70% of dry cell weight. In YEM with L-cysteine medium after 48 h, the PHB accumulated in Rhizobium sp. was more in YEM when inoculated with cysteine. Tajima et al. (2003) isolated PHB-producing Gram-positive bacteria from grassland. The PHB productivity from isolate INT005 was more compared to B. megaterium. Mukhopadhyay and Paul (2003) studied phototrophic purple non-sulfur strain for the potential PHB accumulation. Thirty bacteria were identified by sludge/water sources taken from various areas of east India. Preliminary screening of these microbes in favor of biopolymer accumulation led to recognition of 5 bacteria by amount of biopolymer between approximately 15% dried biomass,

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while cultured on acetate-enriched media with 10,000 lux intensity radiance. Carbon sources such as acetate and butyrate were highly suitable for PHB production. Although nitrogen source in the culture medium showed negative effect on PHB production, the microbial growth was increased. Limiting conditions for phosphate and sulfate increase the production of polymer by the bacteria. The effects of factors like pH and light intensity was also studied in PHB production. Pseudomonas sp. 14–3 (from Antarctic) accumulated poly-β-hydroxybutyrate in high quantity, while cultured upon the media which enriched with octanoate. On the basis of morphological characteristics and rRNA gene sequencing selected bacterium was identified (Ayub et al. 2004). Yilmaz et al. (2005) screened less than 30 Bacillus taken by the grass field in the capital of Turkey; those were characterized like Bacillus licheniformis, Bacillus brevis, Bacillus circulans, Bacillus megaterium, Bacillus subtilis, Bacillus sphaericus, and Bacillus coagulans. Poly-β-hydroxybutyrate accumulation by the above-mentioned isolates was identified through the spectrophotometer and concluded as bioplastic accumulation between 1.06 and 41.67% (w/v) upon the basis of dried biomass. Maximum bioplastic production (41.67% w/v) was found in B. brevis M6. Singh and Parmar (2011) collected 16 unknown samples, analyzed them, and compared with biopolymer-producing strains like Ralstonia, Bacillus, and Pseudomonas. Production of bioplastic by these strains was confirmed with microscopic techniques. This was the first report which demonstrates the synthesis of biopolymer experimentally by two novel isolates like Stenotrophomonas maltophilia and Rahnella aquatilis. Stenotrophomonas maltophilia was isolated from the local beach while Rahnella aquatilis was isolated from a plastic toy. Preethi et al. (2012) isolated bacterial isolate SPY-1 which produced high PHB in comparison with different isolates. The strain had the capacity to accumulate 80% of the dry cell weight as PHB. Hungund et al. (2013) screened soil samples from spent wash and oil mill for the screening of PHB-accumulating strains. One isolate that is Paenibacillus durus accumulates PHB in nitrogen-limiting medium in which fructose was present as the main carbon compound. Singh (2014) studied halophilic and halotolerant microbial strains from industrial and agricultural land. These strains were identified as Bacillus subtilis and Pseudomonas sp. Luria agar medium was identified as the best culture medium for these isolates. Biradar et al. (2015) isolated a total of six bacterial species as PHB producers. Out of them, isolate BBKGBS6 characterized as Lysinibacillus sphaericus BBKGBS6 by 16S rRNA sequencing and produced high amount of PHB.

7.5

Importance of PHB to Bacteria

When carbon is present in excess and phosphorous and nitrogen are available in restricted amount, then poly-beta-hydroxybutyrate granules are present as energyproducing bodies. Senior and Dawes (1971) reported that PHB might act like a reservoir of sinking energy; hence, it is known as reducing–oxidaizing reaction

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regulator material inside microbe. Ensphere of Azotobacter in laboratory was associated with PHB production (Stevenson and Socolofsky 1973). However, in natural environment, storage of high quantity of PHB was not required for encystment formation. Anderson and Dawes (1990) reported microbes with accumulation of biopolymer impeded the debasement of cytosolic parts like protein/RNA during supplement starvation continously, be that as it may, not compulsory always. Poly-betahydroxybutyrate increased the survival of few microorganisms and also was used like an organic and vitality hotspot for spore development in Bacillus sp. Steinbuchel (1991) published that PHB was accumulated like an intracellular cytoplasmic inclusion body in microbial cell. These inclusion bodies act as a reduced biopolymer, given the bit of leeway and those become inaccessible carbon hotspot in favor of contending bacteria, what’s more, that they were osmotically idle, hence influence the osmotic weight of the bacteria. Steinbuchel (1991) announced PHB accumulation in Ralstonia eutropha/Rhodospirillum rubrum contained pyridine of acetoacetyl coenzyme-A. In this manner, biopolymer reservoir for diminishing counterparts and viewed as a fermentated product. In Rhizobium/Bradyrhizobium japonicum, at some stage in the bacteroid form, the N2-assimilating enzymes rivaled by PHB production for reducing counterparts (Povolo et al. 1994; Hamieh et al. 2013).

7.6

Physical and Chemical Properties of PHB

Like any other polymer, chains of monomers in PHB form either homopolymer or heteropolymer. Generally, PHB known to be linear are composed of 3-hydroxybutanoic acid monomer units, each unit from an ester bond with the hydroxyl group of the other one. PHB have an extremely regular structure; all the side gatherings on the polymer chain point a similar way. This makes the chains structure helical structures, with the side gatherings all directing endlessly from the focal point of the helix toward limit steric hindrance. Mass of biopolymer produced generally occurs in between 1  104 and 3  106 g/mol (Doi et al. 1990). PHB is a very low strength biomolecule that need to improve. High molecular weight PHB is valuable for delivering films and solid strands. The molecular weight of PHBs based on PHB-producing enzymes, that different into substrate necessity (Hazer et al. 2012; Kumar et al. 2013). Carbon sources play a key role in diversification of PHB production. These carbon sources include saccharides, n-alkanes, n-alkanoic acids, and n-alcohols. Waste streams like frying oil, vinegar, fats, food, agriculture, wastewater, and oil mill effluents provide free source of carbon for PHB production (Amy Tan et al. 2014). The PHB composition also depends on the metabolic pathway involved. The molecular weights have been measured by gel permeation chromatography, light scattering, and sedimentation analysis. Their monomer composition has been analyzed by mass spectroscopy, gas chromatography, and nuclear magnetic resonance (NMR) detection. PHBs crystalline structure, poly dispersities, enthalpy of

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fusion, melting point, glass transition temperature, and mechanical properties were characterized by the help of various procedures such as differential scanning chromatography. Poly-beta-hydroxybutyrate, compared with the synthetic plastic like polypropylene, is identified as having the most similar properties. Likewise, biopolymer not only remains undissolved in aqueous solution or resistant to hydrolytic decomposition but also gets solubilized in organic compounds like chloroform and other chlorinated hydrocarbons. In the case of PHB inside the bacteria, it presents in a cytoplasm, amorphous state. After extraction from the bacteria, PHB becomes crystalline and rigid but a brittle material. The brittleness of PHB during mechanical processes makes it unresistant to stress (Hazer and Steinbüchel 2007). There is another monomer included in the structure of PHB such as 3 HV (3-hydroxyvalerate) which decreases PHB crystallinity and increases its elasticity (Gross et al. 1989). The glass transition temperature/dissolving temperature of poly-betahydroxybutyrate are around 4  C/180  C correspondingly. The major problem of PHB is that it decomposes near its melting point. The thickness of formless biopolymer is 1.18 g cm 3 and crystalline PHB thickness is 1.26 g cm 3. Augmentation for damage (5%) of poly-beta-hydroxybutyrate is less than expansion for damage of poly-propylene (400%). In general, poly-beta-hydroxybutyrates are hard and weak polymers contrasted with poly-propylene. Weakness is because of the development of enormous translucent spaces as spherulites (Barham and Keller 1986). Super high sub-atomic PHB was set up by modified E. coli with qualities from C. necator. It has higher mechanical properties than regular PHB (Kusaka et al. 1998). PHB has a few properties like poly-propylene with three exceptional highlights: thermo plastic machinability, protection by aqueous solution like water, and complete biodegradability (Page 1995).

7.7

Genes Involved in PHB Biosynthesis

The first PHB genes were isolated from Zoogloea ramigera by Peoples et al. (1987). From that point forward, various qualities associated with the development of PHBs have been described and cloned from different microorganisms. The partition and quality association of pha (gene-encoding proteins for scl and mcl-PHAs) contrasts with different species (Reddy et al. 2003). In the three-stage scl PHA biosynthetic pathway, the genes are known as pha-A (β-ketothiolase), pha-B (aceto-acetyl-CoA reductase), and pha-C (PHA synthase/PHA polymerase). The gene pha-A codes for the compound β-ketoacyl CoA thiolase, which catalyzes the initial phase in the PHB amalgamation. It is an individual from a group of protein associated with the thio-lytic breakage of materials into acyl coenzyme-A and acetyl coenzyme-A. They are found in advanced eukaryotes, yeasts, and prokaryotes and separated in two gatherings dependent upon substrate explicitness. One gathering comprises thiolases expansive explicitness of β-ketoacyl CoA going from 4 to 16 carbon units. These qualities are available in a large portion of the scl

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PHA-delivering creatures and in numerous microscopic organisms pha-B is related with PHA synthase. In R. eutropha, it is situated among pha-A and pha-C qualities and structures the pha-CAB operon (Schubert et al. 1988), while in Bacillus sp. it structures pha-RBC operon, wherein it is flanked by the pha-R and Pha-C (McCool and Cannon 1999). The chemical coded by the pha-B-NADPH-dependent acetoacetyl CoA reductase is an (R)-3-hydroxyacyl CoA dehydrogenase, and it catalyzes the next stage in poly-beta-hydroxybutyrate biosynthesis pathway from changing over aceto-acetyl coenzyme-A in 3-hydroxybutyryl coenzyme-A. Based on the mode of production, subunit arrangement, and material relevence, the PHA synthases are divided into four different classes. Type I PHA synthase is identified from protein of Ralstonia eutropha and mol. weight of the subunits is 64.3 kDa. Pseudomonas group represents the type II PHA synthase. The molecular weight of the PHA synthase subunit is 62.3 kDa. PHA synthase of type III is identified from protein of Chromatium vinosum. It consists of two different subunits coded by two genes pha-C and pha-E exhibiting the molecular weight of 39.7 and 40.5 kDa, respectively. McCool and Cannon (1999) studied polyhydroxyalkanoates synthase of B. megaterium like a distinctive one and measured as more current sort (type IV). The molecular weight of the PHA synthase is identified as 40 kDa and another protein associated with this pha-R protein (22 kDa), coded by the gene pha-R, regulates the PHA synthase activity upon binding. The size of the gene varies from 1 to 2 kb. In Bacillus megaterium, the size of pha-C is 1089 base pairs (McCool and Cannon 1999). The PHA synthase (encoded by Pha-C) uses the coenzyme-A thio-esters of hydroxyalkanoic acids like material; initiate aggregation of various hydroxyalkanoic acids into polyhydroxyalkanoate by discharge of coenzyme-A. In mcl-PHAs, there are additional genes encoding for the intermediary enzymes, namely pha-G (3-hydroxyacyl-acyl carrier protein-coenzyme-A transferase), pha-J (enoyl-CoA hydratase) besides PHA synthase. The pha-G gene was reported like an assimilation connection in between denovo fatty acid biosynthesis and PHB biosynthesis by P. putida KT2440 (Rehm and Kruger 1998). This enzyme converts 3-hydroxy ACP interceders of fatty acid production equivalent to coenzyme-A derivative, resultant 3-hydroxyacyl coenzyme-A as it may make use of like material in support of PHB synthesis. Two Pseudomonas aeruginosa genes homolgous with pha-JAC, assigned like the pha-J1/pha-J2 with different substrate specificities were also cloned, and their role in PHA production was investigated (Tsuge et al. 2000). However, Pseudomonas isolates have two kinds of PHB synthases called as pha-C1/ pha-C2 which vary with material relevance; singular unit arrangement of produced polyhydroxyalkanoates (Matsusaki et al. 1998). Other than the synthesis genes, phaZ is likewise needed to prepare the PHB inclusion bodies from delivering carbon and power once the restricted supplement is re-established (Rehm and Steinbüchel 1999). Umeda et al. (1998) announced that into Alcaligenes latus, Acinetobacter species, Ralstonia eutropha, and P. acidophila, phb CAB genes are paired upon genetic material despite the fact that not really in a similar request. In Rhizobium meliloti, Paracoccus denitrificans, and Z. ramigera, phb-AB and phb-C locus are not related.

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A PHB polymerase has two subunits, Thiocystis violacea, Synechocystis, and Chromatium vinosum determined from pha-E and pha-C (Hein et al. 1998). The PHA biosynthetic genes pha-A (3-ketothiolase), pha-B (NADPH-dependent acetoacetyl coenzyme-A reductase), and pha-C (PHA synthase) by acetyl coenzyme-A are bunched and situated in a gene segment pha-CAB. The locus determining genes in support of PHB accumulation has been portrayed by 18 variable strains (Madison and Huisman 1999). Six genes determining proteins identified with PHB metabolism are bunched in two different loci, pha-ZCPR and pha-AB, in a PHB-accumulating bacterium R. sphaeroides FJ1 isolated from wastewater. PHB synthase, the key enzyme for PHB production, is continuously produced and its production level is not affected by different growth conditions (Yang et al. 2006). In A. caviae, the PHA polymerase gene is flanked by an additional PHA biosynthetic enzyme enoyl-CoA hydratase encoded by pha-J (Fukui and Doi 1997). A few species like P. denitrificans had neighboring PHB synthase more genes as pha-P (determining phasing)/pha-R (determining regulator protein) identified with PHB biosynthesis (Rehm and Steinbüchel 2002). P. oleovorans and P. aeruginosa have two different types of pha-C genes that are differenciated from genes for PHB present inside the cell (Rehm and Steinbüchel 2002). Additionally, down stream of synthase gene preparation, the pha-D gene is collinearly situated, trailed from pha-I and pha-F, those are transcribed into reverse track and determine basic and administrative enzymes (Rehm 2003). Isolates, namely C. vinosum, T. violacea, and Synechocystis sp. possess two subunits of PHB synthase, pha-C and pha-E, which are in one locus yet uniquely situated (Hein et al. 1998). Class IV synthase genes are present in microbes having a place with Bacillus; it involves pha-R and pha-C, those are isolated with pha-B (Rehm 2003). The regulation of PHB genes hushes up complex subject to various elements like accessibility of chemicals, elective variables, two-segment administrative frameworks, and auto inciting particles. In Pseudomonas species, the PHB synthesis locus other than two PHB synthases, pha-C1/pha-C2 isolated by pha-Z is trailed with pha-D, pha-F, and pha-I (Hoffmann and Rehm 2004). It was discovered that P. aeruginosa, P. putida, and P. oleovorans contain three open reading frames (ORF) such as ORF-1, pha-I, and pha-F (Prieto et al. 1999). The consensus sequence like sigma 54 (RpoN) of E. coli promoter upstream of pha-C1 was found in P. aeruginosa and P. oleovorans recommendating which was associated with controlling organization of PHA metabolic reactions into those microorganisms (Timm and Steinbuchel 1992). A pha-C1 promoter part was not as much dynamic when citric acid/glucose is utilized like a carbon compound yet highly dynamic under occurrence with octanoic acid hence confirming the fact that promoter’s initiation relies on the sort of carbon source (Prieto et al. 1999). It was seen into Pseudomonas oleovorans that disruption of pha-F brought more appearance of phaC1 (Prieto et al. 1999). In Acinetobacter sp., PHB production is activated by low phosphate amount as a phosphate inducible promoter was discovered upstream of the initial gene, pha-B (Schembri et al. 1995). The production of PHB in Vibrio harveyi is directed by a β-hydroxybutyryl homoserine lactone which is a molecule for signaling that collects

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at high densities (Sun et al. 1994). Mutation reports in R. eutropha indicate that expanded transition of acetyl-CoA to PHB promotes more production (Park and Lee 1996). Transformation in the phbHI modifies the circumstances of PHB production in R. eutropha proposing the regulatory function for relating enzymes (Madison and Huisman 1999).

7.8

Biodegradation of PHB

Normally, bioplastic decomposition happens generally through scission of the principle chains or side chains of macromolecules, instigated by warm initiation, oxidation, photolysis, radiolysis, and hydrolysis. The character that recognizes PHA by oil-based synthetic plastics is their biodegradability. The biological decomposing of PHA in common habitats (stream and ocean waters, soil, slope, and manure) is one of the economically appealing highlights (Muhammadi et al. 2015). Biological degradation of PHB in the presence of oxygen brings about H2O and CO2, while under the absence of oxygen, the biodegradation compounds are CH4 and CO2. PHB is used by microbes as a vitality source. PHB is biologically degraded in microbial dynamic conditions (Poirier et al. 1995a, b; Lee 1996a, b). Microbes grow upon outer part of a biopolymer and discharge chemicals that decompose PHB into monomers. These monomers are then utilized by the microbes as a carbon source for biomass development. The pace of bioplastic degradation relies upon an assortment of elements containing outside territory, bacterial action of removal condition, acid and base index, heat, dampness, and occurrence of various supplements. PHB is not able to dissolve in water or isn’t influenced by dampness. It doesn’t decompose under ordinary states of capacity and is steady inconclusively in air (Mergaert et al. 1992). Kim et al. (1997) saw that microbial for the most part surpassed the bacterial biomass and accordingly almost certainly growths may assume an impressive function in debasing polyesters, similarly as they dominatingly play out the deterioration of natural material in the environment. Biodegradation of PHB was described by the “soil burial experiment.” Indicated reduction in the weight of biopolymers covered inside earth surface to 1–2 months was 8.5–57.3% at 28  C; 11.6–86.7% at 37  C or afterward diminished up to 7.2–25.9% at 60  C. PHB decomposed highly at 37  C than at 28  C and 60  C. Practically identical outcomes have been watched already (Kim 2000). PHB has been accounted in the direction of decomposition into oceanic conditions (Lake Lugano, Switzerland) in 8 months at 6  C temperature (Johnstone 1990).

7.9

Identification of PHA by Staining Techniques

Whereas confining PHA-producing microorganisms by enviroment, that is important for monitoring quickly large assortment of microbes under brief timeframe. Dyes explicit for PHA are utilized inside location to inclusion bodies. Practical

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province recoloring strategy has been recommended like technique for quick viewing of PHA-aggregating microorganisms. Hartman (1940) was pioneer for recommended utilization of Sudan Black-B for microbial lipid staining. Therefore, Burdon et al. (1942) affirmed more noteworthy estimation with stain and adjusted system to show intracellular greasy compound inside microorganisms from getting ready glass slides of microscopic organisms recolored by alcoholic Sudan Black-B stain further counterstained by red colored dye, safranin. A method to exhibit intracellular greasy compound inside microorganisms with Sudan Black-B stain and the desiccated slide counterstained from red colored dye was given by Lewis (1941). Ostle and Holt (1982) announced that poly-betahydroxybutyrate bodies displayed solid orangish fluorescence while recolored from Nileblue-A. Warmth preset isolates were stained by Nileblue-A after that haste was seen at 460 nm. The culture screening or determination framework was depicted to investigate the accumulation of PHB in R. capsulatus (Kranz et al. 1997). Screening by Nile red disintegrated in (CH3)2CO recognized PHB-producing and non-producing strains. Spiekerman et al. (1999) suggested the utilization of a delicate feasible colony recoloring strategy utilizing Nile red used to view microorganisms to store PHB by naked eyes. An immediate consideration of stain into media didn’t influence the development of microbes yet permitted to locate occurrence of PHB into the feasible cultures whenever throughout development. These PHB-producing microorganisms showed intense fluorescence while seen in ultraviolet rays. Juan et al. (1998) utilized reasonable culture view strategy for quick identification and screening of poly-betahydroxybutyrate accumulating exopolysaccharide lacking transformates by parental R. meliloti. Alcoholic Sudan Black-B was poured for recoloring microbial culture that was developed upon solid Luria Bertani media and culture was saved untouched for 30 min atleast. Stain was tapped and culture was delicately flushed with absolute alcohol. Bacterial culture unfit for absorbing Sudan Black-B remained uncolored, whereas polyhydroxybutyrate-accumulating strains seemed somewhat dark blue. Phosphine R is a fluorescent stain of PHB identification; in this technique, bacterial colonies are grown on agar plates in the presence of phosphine 3R which is a lipophylic dye. As indicated by this recoloring, the fluorescence of colonies under UV rays shows the presence of the biopolymer. Microbial colonies with high amount of PHB produce bright-green colored fluorescence under UV rays (Bonartseva and Myshkina 1985). Hamieh et al. (2013) detected the PHB in bacterial cell by transmission electron microscopy.

7.10

PHB Extraction and Recovery

PHB is an intracellular compound, which shows up as incorporation bodies in microorganisms (Kapritchkoff et al. 2006). In this manner, if exceptionally clean PHB is expected, cell rupturing ought to be done to break the cell membrame and secrete PHB. As such, extraction of biopolymer from the microbes is an essential

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phase of the cycle (Anderson and Dawes 1990) and polymer recuperation and cleaning steps are significant commitments to the creation price (Kapritchkoff et al. 2006). Biopolymer recovery steps, notwithstanding the expenses of keeping up unadulterated colonies and significant expenses of natural materials, are one more thing which adds toward significant expense of PHB production. Within a span of previous 20 years, a few recovery measures have been studied and researched so as to locate the monetary method to confine and decontaminate PHB. PHB is an intracellular product, a number of solvents have been studied for PHB extraction with the aim of finding a relatively cheaper method while achieving higher rate of recovery. Doi et al. (1990) portrayed chloroform recovery strategy. PHB was recovered with heated chloroform into soxhlet to longer than 60 min. At that point, PHB recovered was isolated by fat from hastening through hexane, diethyl ether, and alcohol. At long last, PHB was redisintegrated in chloroform and additional cleansing done by precipitation with hexane. Ramsay et al. (1994) inspected extraction of PHB by three distinctive chlorinerich solutions like chloroform, 1,2-dichloroethane, and methylene chloride. They got the best recovery and purity when microbial cells were pretreated with (CH3)2CO. For all the three solvents, 15 min were ideal digestion time. Extended treatment brought decomposition of PHB weight. A level of extraction after the cells were pretreated with (CH3)2CO were 70, 24, and 66%, while refluxed to 15 min by chloroform, methylene chloride, and 1,2-dichloroethane individually. Though degree of cleanliness of those three solutions into ideal situations were 96, 95, and 93%, separately. Ramsay et al. (1990) analyzed PHB extraction measure by Ralstonia eutropha utilizing hypochlorite treatment by detergent. Triton X100 and sodium dodecyl sulfate were examined those act as detergent in the reaction. An increase in cleanliness and molecular weight could be gotten from pretreating by detergent before the recovery from sodium hypochlorite. They revealed that detergent separated eliminated from absolute protein and extra enzyme was additionally taken out from sodium hypochlorite treatment. Fidler and Dennis (1992) revealed framework for extraction of biopolymer bodies from Escherichia coli with communicating T7 bacteriophage genes. The lysozyme infiltrated damages the cell membrane being the reason for the release of PHB inclusion bodies. The framework created utilized different extra chromosomal plasmid and communicated at lower point all through the cell division process. Toward finish of collection phase, biomass were reaped then resuspended into sequestering compound. That actuated lysozyme to damage cellular membrane and deliver PHB when aggregation arrived at the most extreme. Triton X100 was additionally supplemented for the help in the cellular membrane disintegration. The productivity of rupturing of membrane was more noteworthy than 99% detailed by them. Hahn et al. (1994) suggested strategy known as scattering by sodium hypochlorite and chloroform. They guaranteed about technique eliminated the greater part of the non-PHB cell materials during sodium hypochlorite treatment, which encouraged the division of PHB from the microbes. Treatment with sodium hypochlorite diminished the thickness of the chloroform stage. They likewise researched on the

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ideal state for PHB extraction from R. eutropha utilizing scatterings of chloroform and sodium hypochlorite. The ideal parameters from their trails were accounted for to be 90 min assimilation time with 30% solution of sodium hypochlorite and chloroform to watery phase proportion of 1:1 in volume. They acquired level of extraction of 91% and the degree of virtue more compared to 97%. The pace of decomposition expanded like the amount of sodium hypochlorite expanded. Because of the significant expense of dissolvable extraction, the proteolytic assimilation strategy was created via ICI, UK. Parts of that cycle incorporated heat action at 100–150  C to break the membrane and deform genetic material; proteolytic processing and washing with positively charged detergent to cell particles without PHB. At last, concentrated PHB from centrifugation was treated with H2O2. Now, PHB could be isolated from the liquid with the help of sedimentation technique. A serious damage of PHB during sodium hypochlorite treatment was every now and then announced. A decrease in molecular weight up to 50% of the PHB was reported when the microbial cells were treated with sodium hypochlorite (Lee 1996a, b). As indicated by Steinbuchel (1996), ICI utilized a blend of different enzymatic proteins during the enzymatic processing stage. These catalysts were phospholipase, lysozyme, proteinase, lecithinase, etc. These biological catalysts hydrolyzed a large portion of biomass except PHB but PHB stayed unaffected. Braunegg et al. (1998) detailed that ICI later utilized proteolytic compounds like pepsin, trypsin, and papain and a blend of these catalysts to extract PHB. PHB recovers from the chloroform phase by non-solvent (aqueous solution of methanol) precipitation and filtration, under ideal parameters, the extraction was up to 91% and the purity of extracted PHB was more than 97%. Shawaphun and Manangan, 2009, reported the extraction of PHB produced by A. latus and found that CHCl3 and CH2Cl2 gave relatively higher rates of PHB recovery (83.93% and 86.80%) compared to other non-solvents and protic solvents like toluene and hexane. Bacillus sp. isolates CL1 and Ralstonia eutropha, enclosed PHB inside the microbial cytoplasm and phasing gene mutant R. eutropha having a solitary big PHB bodies. The optimum PHB bodies are encompassed by phosphor-lipids film and glass-like protein. Concentrated cell mass is dried either by freeze or spraydrying. PHB is recovered from untreated cell mass by dissolving it in a natural dissolvable, generally chloroform, chloro-limited methylene chloride, propylene carbonate, and dichloroethane (Charen et al. 2014; Gomaa 2014). Subsequent to eliminating cell parts by filtration and sedimentation, the PHB is sedimented in solvents that cannot dissolve PHB, for example, cool alcohol (Riedel et al. 2013). Solvents recover the PHB without damaging it by enhancing the cell membrane porousness of the PHB (Kunasundari and Sudesh 2011). Yang et al. (2011) built up a system for PHB utilizing straight alkyl benzene sulfonic acid like an option in contrast for regularly utilizing sodium dodecyl. In this technique, just 21% of the detergent is neccesary, compared to past techniques. Singh et al. (2009) looked into a novel self lyse microbial cell for extraction of PHB accumulated into Bacillus megaterium. Here framework, a gene group conveying the membrane rupturing and endo lysin of Bacillus amyloliquefaciens phage (Morita et al. 2001) was

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embedded in Escherichia coli and Bacillus subtilis transport vector pX. Herein expression scheme, “xyl R-xyl A” aimed genes are incited by xylose however hindered by dextrose (Salzberg and Helmann 2007). This contemporises cycle of unconstrained membrane degradation and material depletion, and this brings about an arrival of amassed PHB.

7.11

Growth Parameters to Increase PHB Production

Suzuki et al. (1986) detailed that Pseudomonas sp. developed into alcohol like only carbon and vitality supplement had delivered approximately 65% of PHB upon the basis of dried biomass measurement. So as to get more amount of bioplastic, legitimate media synthesis was used. Here, the amount of phosphate and ammonium were kept up at lower side. Nitrogen inadequacy was discovered as the best method for invigorate production of bioplastic. The restriction of soluble O2 amount was identified to diminish pace of cell mass development and PHB accumulation. This result was opposing outcomes announced from different researchers. Sillman and Casida (1986) deliberate vesicle development against PHB production into Azotobacter vinelandii ATCC12837. While inoculated into Burks agar with 0.2 mg of glucose per ml that produced bioplastic before vesicle development. Expansion of low degrees of ammonium chloride, urea/adenine caused an addition in vesicle development but PHB development was diminished. Hypo-xanthine and inosine expansion likewise invigorated vesicle development, despite the fact that PHB development was not decreased. It was reasoned that vesicle development by Azotobacter didn’t need production higher than minimum concentration of PHB. Poul et al. (1990) contemplated an impact with various carbon compounds at C: N proportion as 15:1. PHB collection was important in extended log phase to fixed period of development with noteworthy amounts of bioplastic produced upon malate like contrasted to acetic acid derivation, pyruvate, lactate, and fructose. More elevated stages were identified to gather while low PO2 conditions were kept up through development. As per Doi et al. (1990), two phases of fed-batch culture technique are generally utilized procedure to augment large amount of biomass and inclusion bodies. In the beginning of development stage, ideal wholesome conditions were utilized to build up a high cell number. At that point, a chosen supplement was restricted to invigorate PHB accumulation in the subsequent stage or production stage. Daniel et al. (1992) detailed so as to Pseudomonas strain 135 while developed into ammonium restricted fed-batch culture utilizing alcohol like only carbon and vitality compound; this could accumulate the highest biopolymer that is 55%. At the point when the microbes were cultured on magnesium and phosphate-restricted medium, the accumulation of PHB were approximately 40% and 35%, separately. Kim et al. (1994) detailed as a fed-batch culture of R. eutropha strain NCIMB 11599 by controlled sugar amount and nitrogen constraint offers 121 g/L of PHB accumulation. At the point while nitrogen restriction condition was utilized to instigate bioplastic production, sodium hydroxide was added to the media to maintain acid-

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base equillibrium. Although, because of the cell membrane rupturing brought about by the harmfulness of sodium hydroxide, more cellular aging density was inconceivable. Moreover, this was essential to keep up phosphate and magnesium concentrations more than 0.35 g/L and 10 mg/L individually (Asenjo et al. 1995). Ryu et al. (1997) as a result subsequently embraced phosphate restriction methodology to incite bioplastic production where pH was constrained from expansion of ammonium hydroxide. In these parameters, 232 g/L of bioplastic were acquired. Shimizu et al. (1994) researched accumulation of PHB by Ralstonia eutropha H16 fed in the company of butyric acid and valeric acid. Ideal parameters used for biopolymer accumulation were 3 g/L butyric acid at pH 8.0 by these bacteria. Seventy five percent of PHB was acquired beneath these parameters, whereas lesser biopolymer was produced once pH was set at 8.0. At this point when the amount of butyric acid was set at 0.03, 0.3, and 10 g/l, accumulation of biopolymer was 44%, 55%, and 63%, individually produced. Bourque et al. (1995) researched the polyhydroxybutyrate accumulation by Methylobacterium extorquens ATCC 55366 utilizing methanol like chief carbon and vitality compound into a fed-batch fermentation technique. Polyhydroxybutyrate accumulation somewhere in the range of 40 and 46% on the basis of dried weight was formed by M. extorquens. Moreover, biomass synthesis and development pace of M. extorquens were influenced from minerals provided in different combinations. Biomass synthesis and the specific development pace of this bacteria was negatively affected in the absence of FeSO4, CaCl2, (NH4)2SO4, MnSO4, and ZnSO4. Parshad et al. (2001) checked for 37 strains and transformates of Azotobacter chroococcum in favor of polyhydroxybutyrate accumulation utilizing Sudan BlackB stain. Maximum PHB production was found to be with 2% glucose and 15 mM/L ammonium salt on 36 and 48 h of development into submerged culturing and into immobile culturing individually. They additionally saw that the purpose of polyhydroxybutyrate accumulation was elevated upon sucrose and D-glucose. Among inorganic nitrogen compounds, they discovered 15 mM/L ammonium acetic acid derivative concentration to be the best for polyhydroxybutyrate accumulation. Ghatnekar et al. (2002) examined polyhydroxybutyrate accumulation by Methylobacterium SPV-49. They assessed various carbon compounds. Most extreme production of polyhydroxybutyrate was seen among glucose like the carbon nutrient. Alcohol with saccharides like lactose and sucrose were also additionally stimutaled PHB production. The impact of carbon to nitrogen proportion on PHB production was analyzed. Various procedures for recovery and isolation of the PHB from microbes were studied. The non-dissolvable-based strategy utilizing an elevated stress homogenizer within sight of sodium dodecyle sulfate was discovered to be generally good. Highest isolation of 98% was accomplished by homogenizing the biomass at 400 kg cm 2 in 5% sodium dodecyle sulfate. Mahishi et al. (2003) examined the enhancement of polyhydroxybutyrate accumulation from transgenic E. coli holding the polyhydroxybutyrate incorporating genes Pha-CSa and Pha-BSa from S. aureofaciens NRRL 2209. Impacts of various C and N compounds on polymer production from transgenic E. coli were examined.

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Among the carbon compounds utilized, palm oil, glucose, glycerol, and ethanol upheld PHB production. No polymer production was detected into genetically modified bacteria while sugarcane molasses was utilized like a carbon supply. A mixture of yeast extract, peptone, and corn-steep alcohol was utilized like nitrogen supply. The highest production (60%) of microbial dried biomass was estimated at 48 h of culture development at 37  C into media containing glycerol as the chief carbon nutrient and yeast extract-peptone mixture like the nitrogen resource. Khanna and Srivastava (2005) improved cultivation conditions intended for the development of Ralstonia eutropha NRRL814690 within sight of supplements to diminish the extraction price of PHB. Along with fructose and (NH4)2SO4 like carbon and nitrogen compounds individually, Ralstonia eutropha showed highest cell mass of 3.25 g/L with polyhydroxubutyrate amount 1.4 g/L within 2 days. To decide the chances of development capability of Ralstonia eutropha, this was developed into various carbon compounds, of which lactic acid, fructose, glucose, and sucrose yielded great biomass formed and polyhydroxubutyrate granules. So as to fuse less expensive N compounds and developmental factors in culture medium, (NH4)2SO4 was replaced by (NH4)2NO3, urea, and NH4Cl. The highest PHB production 3.84 g/L after 60 h of incubation obtained with urea as nitrogen source. Senthil and Prabhakaran (2006) researched an impact of pH on production of polyhydroxubutyrate by Alcaligenes eutrophus MTCC1285 in various materials. They examined polyhydroxubutyrate accumulation at pH as 6.9 and 8.0. The concentration of polyhydroxubutyrate accumulation at pH 6.9 was 0.8, 0.5, 0.4, and at pH 8.0, they were 1.1, 0.65, 0.55, and 1.0 g/mL in sago, thippi, glucose, and molasses material containing culture medium, individually. Koller et al. (2007) reported the optimization of polymer accumulation on Hydrogenophaga pseudoflava by using hydrolyzed whey and valerate as substrate. Haas et al. (2008) studied the use of potato saccharide, saccharified waste like carbon compound for increased PHB production for Cupriavidus necator. Cavalheiro et al. (2009) evaluated that the molecular weight of PHB accumulated with C. necator by glycerol in a range of 7.86  102 kDa (used glycerol) and 9.57  102 kDa (fresh glycerol) that permits preparing from basic methods of the plastic business. Chee et al. (2010) screened the effect of lauric acid, myristic acid, oleic acid, palmitic acid, and stearic acid on Burkholderia sp. USM. Nikodinovic Runic et al. (2011) reported that Pseudomonas putida produced mcl-PHA with styrene as substrate. Zafar et al. (2012) reported P(3HB) production using A. lata. In this investigation, the enhancement of PHB accumulation from A. lata MTCC 2311 was completed. From utilizing the hereditary algorithm upon the artificial neural organization, the anticipated most extreme PHB creation of 5.95 g/L originated with 35.2 g/L of sucrose and 1.58 g/L of urea; in any case, the most noteworthy trial PHB 5.25 g/L was accomplished utilizing 36.48 g/L of sucrose. Povolo et al. (2013) screened lactose and sucrose as carbon source for PHB production on Hydrogenophaga pseudoflava, which showed PHB yield of 20.2–62.5% (w/w). Mozumder et al. (2015) investigated impure glycerol for its

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likely use as a modest feedstock for PHB production. The highest cell mass amount of 104.7 g/L was achieved with a PHB into the liquid cultural medium of 65.6 g/L.

7.12

Factors Affecting PHB Production

A few variables should be considered in the choice of microbes for the PHB production at industrial level for example, the capacity of microorganism to use a reasonable carbon compound, development pace, PHB accumulation intensity and the most extreme degree of PHB production (Ojumu et al. 2004). Different strategies are being developed to enhance the PHB accumulation and to reduce the complete price.

7.12.1 Microorganisms The main view to consider is the compatibility of the microorganism for systematic accumulation of PHB by inexpensive sources (Keshavarz and Roy 2010) by balancing the limiting conditions required for PHB production. Enhancing the production rates of high yielding PHB cultures is being done. Faster growth rate and developing copolymers blends by using engineering recombinant microbes such as E. coli and P. oleovorans is also being investigated (Reddy et al. 2003; Suriyamongkol et al. 2007; Preusting et al. 1993).

7.12.2 Medium The right selection of medium is necessary for high volumetric productivity of PHB. Choice of medium ingredients is responsible for production of homopolymers or copolymers. Several media from cheap sources to reduce the overall cost such as black treacle (Solaiman et al. 2006), corn-steep alcohol (Nikel et al. 2006), whey (Marangoni et al. 2002; Koller et al. 2008), rice and wheat-bran (Van-Thuoc et al. 2008; Huang et al. 2006), starch and starchy wastewaters (Kim and Chang 1998; Quillaguamán et al. 2005; Halami 2008; Haas et al. 2008), outflow from palm and olive oil mill (Pozo et al. 2002; Bhubalan et al. 2008; Ribera et al. 2001), activated sludge (Yan et al. 2006; Jiang et al. 2009), and swine waste (Cho et al. 1997) are being studied extensively. Starting media pH is important for the cell development and PHB production from C. necator (CCUG52238T) enhancing the underlying media pH at time spans of 0.5 units influenced both the biomass and PHB development. Both the biomass and PHB amount were enhanced when the starting media pH was enchanced from pH 6.0 to 7.0, that is, from 6.42 to 8.57 g/L for biomass and 20% (w/w) to 34% (w/w) to PHB amount, separately (Zahari et al. 2012).

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7.12.3 Fermentation A few techniques, for example, fed-batch and continuous culture method have been completed to enchance efficiency for PHB production (Doi et al. 1990; Lee 1996a, b; Du et al. 2001; Yu and Wang 2001; Du and Yu 2002). Different carbon sources (Durner et al. 2001), limiting nutrients, different feeding regimes (Sun et al. 2007), and different dilution rates (Durner et al. 2000) have been studied. Introduction of blend cultural medium, for example, activated sludge (Satoh et al. 1999; Chua et al. 2003; Lemos et al. 2006) can add and diminish the expense of bioplastics. Feast/ famine sort of fermentation technique has additionally been researched (Dias et al. 2005; Lemos et al. 2006). The best and capable factors integrated shaking rate with confidence 90.04%, trailed by ferrous sulfate, sucrose, and sodium nitrate with certainty stages 81.16, 78.88, and 77.27%, individually (El-Raheem et al. 2013). Solid-state fermentation has been reconsidered like an option to submerged fermentation and could be a potential system in favor of the unexpansive extraction of PHB. The resources venture to solid-state fermentation is generally inferior compared to submerged fermentation and the expense of crude substances for solid-state fermentation would be modest since it utilizes dissipate farming substrates. These positive components make solid-state fermentation an expected procedure for PHB isolation (Sindhu et al. 2015).

7.12.4 Recovery Since extraction of PHB contributes essentially to the general financial aspects of PHB production, a number of processes have been tried to increase PHB recovery. Pretreatment of cells, cell membrane lysis, enzymatic treatment of non-PHB in the framework, unconstrained freedom of PHB, disintegrated air buoyancy, and extraction utilizing supercritical CO2 have been tried (Jacquel et al. 2008).

7.13

PHB Quantification and Characterization

Different methods are available for determining the PHB content, the structure of PHB, and the composition of monomer. The first method that was developed by Law and Slepecky (1961) involved transformation of the PHB to unsaturated carboxylic acid by heat treatment with concentrated sulfuric acid and analyzing spectrophotometrically. This method was superseded by gas chromatography (GC) and highpressure liquid chromatography (HPLC) with a number of variations in sample processing. Braunegg et al. (1978) treated the cells to gentle methano lysis after that checked with gas chromatography. Riis and Mai (1988) improved this method by using propanol and HCl. Chloroform-extracted PHB cells from B. megaterium were quantitated by HCl acid ethanolysis and GC-mass spectrometric (MS) (Findlay and White 1983; Tan et al. 2014). Karr et al. (1983) used ion exclusion HPLC. Brandl et al. (1989) identified amount of PHB with gas chromatography after lysis

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with methanol freeze-dried microbe. After GC analysis, the presence and amount of 3-hydroxybutyric acid were determined. Gel permeation chromatography (GPC), differential scanning calorimeter (DSC), rheology scanning, phase contrast and transmission electron microscopy (TEM) are applied to do the PHB characterization (Yezza et al. 2007; Pereira et al. 2008), GPC for molecular weight measurement, DSC and rheology for melting temperature measurement, and TEM for inclusion body observation. Nuclear magnetic resonance (NMR) technique has also been applied to characterize PHB. Doi et al. (1986) has analyzed the conformation of PHB with 500-MHz 1 H nuclear magnetic resonance spectroscopy and sequence determination of the monomeric units in 3HB-co-3 HV copolymer by analysis of 125-MHz 13C NMR spectra. A number of NMR studies for different PHB to determine the constituent monomers have been done (Rozsa et al. 1996; De Waard et al. 1993; Doi et al. 1995). It has also showed that fourier transform infrared spectroscopy (FTIR) can be used for determining the crystallinity of polymers. Jarute et al. (2004) and Randriamahefa et al. (2003) applied FTIR to check the amount of the PHB in microbial solution (De Rooy et al. 2007). Thin layer chromatography was used to determine the presence of PHB in production media. Black color bands were observed in TLC which indicated the presence of PHB in the production medium (Sharmila et al. 2011).

7.14

Mutagenesis

Transformation in genes of interest assumes significant role enhancing in bioplastic accumulation. Improvement of bacteria by transformation is extremely financially a savvy technique and deliberately a better methodology for gaining highest yield of polymer. Because of moderate development and less PHB amount, the industrial extraction was moved to Alcaligenes eutropus, a table sugar using transformates by first H16 strain (Byrom 1992). ICI side compounds, the UK created PHB valverate on a commercial level from sugar using transformates of R. eutropha by a glucose or propionic acid substrate blend like C compound and in phosphate-restricting state (Steinbuchel 1991). Park and Lee (1996) had the option to plan PHB high accumulating from chemical genetic transformation. They utilized N-methyl N´-nitroso guanidine for constraint of transformant bacteria by changes in enzymes associated with tricarboxylic acid cycle. Isocitrate dehydrogenase mutant of C. necator was screened after genetic transformation. The biopolymer production was essentially expanded because of the enhanced progression of carbon source to the bioplastic biological production cycle rather than the tricarboxylic acid cycle because of the initiation of biopolymer biosynthesis-associated enzymes promoted by the acetyl coenzyme-A collected inside the cell. Katircioglu et al. (2003) determined the accepted mutant of Bacillus megaterium Y6, B. sphaericus X3, B. subtilis K8, and B. firmus G2 were checked for their poly-β-hydroxybutyrate accumulation limits. fadA mutant of E.coli strain WA101 showed increased accumulation of 3-hydroxydecanoate monomers up to 93% from

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sodium decanoate (Park et al. 2002a, b). Aceto-acetyl coenzyme-A reductase and β-ketothiolase were highly expressed in the CydR transformate of Azotobacter vinelandii prompting toward PHB production through the exponential development stage, dissimilar to the original strain that solitarily produced PHB during stationary stage (Wu et al. 2001). It has been indicated that transformations that expand the degree of acetyl coenzyme-A act to enhance the production of bipolymer. The presence of a genetic change in strain may help clarify the decline in biopolymer production at 360 h, anyway more noteworthy exploration is important to verify or refute this hypothesis (Verlinden et al. 2007). Adwitiya et al. (2009) in this investigation assessed the polybeta-hydroxybutyrate synthesis limit selected tranasformation of Bacillus thuringiensis IAM 12077. A Bacillus sp. distinguished like Bacillus thuringiensis IAM 12077 fit to produce 10–15% dry biomass weight poly–beta-hydroxybutyrate when cultured in supplement stock for 48 h was exposed to arbitrary mutagenesis. Within the ultra violate mutant selected, 19 accepted mutants synthesied higher poly–beta-hydroxybutyrate than the original strain, rather 2 bacterial strains synthesized less. B8 mutant showed promising poly-beta-hydroxybutyrate production as 24.68%; 1.54 times with high poly–beta-hydroxybutyrate accumulation that is 1.3 g/L; 5.4 times after that the parental isolate. Chemical transformation produced reputed transformates, of which 6 had a reduction, 3 had no change, and 2 had an enhancement into poly-beta-hydroxybutyrate accumulation. Although the enhancement into production was analogous to enhancement exhibited with the ultraviolet transformate B8, the yields didn’t associatively increment. Some mutants of R. eutropha such as ATCC 17697 have been reported to use sucrose to accumulate PHB (El-Sayed et al. 2009; Abdelhad et al. 2009; Tanamool et al. 2009), little research has been done to prove that mutants of R. eutropha can produce PHB from sucrose as efficiently as with glucose or fructose. Chen et al. (2011) constructed a series of strains and mutants which were able to produce the PHB in a modified M9 medium supplemented with 20 g/L xylose. Santanu et al. (2011) utilized chemical genetic transformation to make transformate of C. necator over producing PHB on cheap carbon compounds. They isolated 5 mutants over producing PHB at minimal-salt media when fructose is used like a carbon compound. Out of them transformates of some were capable of using sugar syrup for more PHB production. Mutations were stimulated with ultraviolet rays, acri-flavin, and 5-bromo urasil. With every bacterium, production of poly-betahydroxybutyrate according to dried biomass weight in percentage was identified in a scale of 1.46–63.45%. Acri-flavin created transformation produced on variable strain that is 173A2 that accumulate 200 μg/ml PHB in 24 h of growth utilizing the supplemented cultural media with 1.5% glycerol as carbon compound (Elsayed et al. 2013). Girdhar et al. (2014) studied the transformation that was done with physical and chemical techniques such as UV light used as physical method and ethyl methyl sulfonate treatment as chemical method on Bacillus flexus and Bacillus megaterium. The outcomes uncovered that there was a magnificent improvement in biopolymer synthesis with right around two folds, when bacteria treatment performed with

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ultraviolet light as physical mutagen; however, no such improvement was seen with chemical mutagen like ethyl methyl sulfonate. The biopolymer production enhanced particularly from 15.68 to 18.61% with chemical mutagen while with ultraviolet light treatment the biopolymer production enhanced exceptionally, from 15.68 to 23.70% in case of Bacillus flexus.

7.15

Future of PHB

As petroleum cost keeps on raising, the world is being compelled to utilize bioplast for numerous uses. Endeavors have been made to utilize PHB in horticultural, clinical, and in bundling businesses. The headways have been found in the regions of bioplastic like conveyance vehicles for controlled medication delivery and advancement of restorative gadgets including inserts and three-dimensional platforms for tissue building (Lei et al. 2017). In fact, recently their functionality in newer areas like producing intracellular nanostructures has made many companies realize and harness their commercial potential for clinical applications, for example, drug conveyance or antibody advancement. An appealing thought is this utilization of reused bioplastic is a practical alternative for 3D printing, in addition to effectively certain highlights of these innovations, for example, reserve funds on crude materials, low vitality cost, just as low CO2 emissions (Lanzotti et al. 2019). Right now their creation is costly; however, these plastics are just in their first phase of business advancement (Lee 1996a, b). An immense body of research has been done in the past 80 years on PHB. Incredible progress have been done into enzyme designing of PHB synthases to change its specificities in order to deliver PHB with wanted monomer arrangement (Suriyamongkol et al. 2007). Wider usage of these composites and blends has allowed overcoming or minimizing the shortcoming in properties that limit their application (Keshavarz and Roy 2010). One of the most promising approaches in overcoming the major drawback of high production cost is the atomic rearing of genetically modified plants communicating practically dynamic PHB biosynthesis pathways and to synthesize PHB legitimately by agribusiness (Steinbuchel and Fuchtenbush 1998); Poirier et al. 1995a, b). Producing bioplastic into crops, in any case, will probably be more costly (3–4 US$/kg) than delivering corn-starch or soyabean-oil, that priced into 0.25–0.50 US$/kg (Suriyamongkol et al. 2007). Also the extraction procedure and processing from plants still need to be researched. In addition, production of PHB from cheap resources, correct balance of ingredients, potential of different bacterial species needs to be investigated further in setting of expanded PHB production and efficiency, various kinds of PHB and simplicity of bioplastic extraction (Quillaguamán et al. 2010). Artifical synthesis or modification of PHB molecules will without a doubt flourish with the gigantic organic assorted variety of character, where identical enzyme exercises can be gotten by extraordinary spots, whereas genetic modifications turn out to be less and to a lesser extent an innovative obstacle (Madison and Huisman 1999). The change of CO2 infers a decrease of ozone harming substance outflows. Hydrogen oxidizing

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microscopic organisms, for example such as Cupriavidus necator can store PHB utilizing CO2 as a carbon source, for example, through an auto-trophic transformation (Mozumder et al. 2015). Notwithstanding, because of its moderate crystallization, thin handling temperature range and inclination to “creep,” it isn’t appealing for some, applications, requiring advancement so as to defeat these weaknesses (Babu et al. 2013).

7.16

Conclusion

In the twenty-first century, we are living in huge load of pollutions from many sources including polythene wastes. Plastic production also involves a number of harmful chemicals which pose environmental as well as human health risks. Natural mechanisms for self-regeneration cannot manage such harmful pollutants. Thus, this chapter has provided important information regarding the optimum parameters for biopolymer accumulation which may take advantage at commercial stage to biopolymer accumulation of a rapidly rising substitute for oil-based artificial polymer.

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Insight of Biopolymers and Applications of Polyhydroxyalkanoates Rishabh Agrahari, Gargi Sarraf, Naveen Chandra Joshi, Swati Mohapatra, and Ajit Varma

Abstract

In this polluted era while we are talking about eco-friendly environment biological matter comes to our mind. Hence, if we go through the available works and copyrights, there will be no exact authentication on the definitions: “degradable,” “biodegradable,” “bio-based,” “compostable,” and “biopolymer”; these words often have overlapping way of explanation and multiple meanings. All these types of biopolymers having different properties and diversified applications. As these biopolymer is the need of the hour to make our environment ecofriendly. The only polymer that carries all the properties of the available properties of the polymer is Polyhydroxyalkanoates. So here in this chapter, we have complied the types and diversities of polymer and highlighted the application of most efficient bio-based, biodegradable, biopolymer “Polyhydroxyalkanoates.”

8.1

Introduction

The intention of the environmental science is the polluted material like plastic should be degraded easily. The term “degradable” can be used for a polymer or plastic that disintegrates into other forms and structured by several physicochemical and biological process. Here, the term defines the ability to degrade not necessarily by a biological mechanism. “Biodegradable” is a term used for polymers that have the properties of degradation using microbes or microbial-derived metabolites under certain environmental, physical, and chemical conditions. However, the term

R. Agrahari · G. Sarraf · N. C. Joshi · S. Mohapatra (*) · A. Varma Amity Institute of Microbial Technology, Amity University, Uttar Pradesh, Noida, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_8

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“biodegradable” is ambiguous on its own. According to the American Society for Testing and Materials standard (ASTM D5488-94de1), the degradation process will be proper if the plastic will be degraded by certain metabolites of the microbes by enzyme action that leads to release of CH4, CO2, H2O, and other inorganic compounds. In other countries, the terms are described differently, for example, the term “Biodegradability” is defined by the Japan BioPlastics Association (JBPA); JBPA explained it as a characteristic of any material capable of undergoing biological disintegration releasing the final product of CO2 and H2O, hence recycled in nature. The terms “Biodegradability” and “Disintegration” share an overlapping meaning but have distinguished meaning; disintegration is used for the characteristics of a material to break into separate and small pieces. When it comes to determining the bioplastics as biodegradable, the pre-established criteria of the ISO method are used for evaluation. To be classified as GreenPla®, the biodegradable plastics must meet the criteria like the content of safe intermediate reaction (“Japan BioPlastics Association (JBPA), Green- Pla®” n.d.). There are some other international standards that classify biodegradable polymers, for example, EN 13432:2000, ISO 17088:2012, ASTM D6400–12, EN 14995:2006. The term “BIO-based” focuses on the basis of organic raw material, especially the polymeric material should be generated from regenerated resource via biological processes or natural bases and end up with CO2, H2O. The raw material is defined as renewable when it comes from a natural process and has its yield equivalent or more than the consumption rate. Similarly, in this context Farm Security and Rural Investment Act (FSRIA) of 2002 defines bio-based product means “commercial or industrial things” apart from food and feed on biologically derived stuffs such as forest litter, kitchen waste, domestic organic waste, or marine materials. Carbon-14 can mostly be detected in a bio-based material very rare in case of fossils, this characteristic represents its half-life of 5700 years. To ensure it, the characteristic of carbon from biomass as raw material is exploited to determine the content of a biopolymer; by quantifying carbon atoms released from a short CO2 cycle (Mohapatra et al. 2017b). Few experiments of 14C suggested, “bio-based materials” can be defined as the degradable organic material derived from non-fossil biological bases. Using liquid scintillation calculating rate of degradation in dpm/g C (disintegrations/minute/ gm carbon) process is measured which can determine the 14C level. A bio-based polyethylene terephthalate (PET) contains nearabout 0.1 dpm/g C of 14C. ASTM D6002-96(2002)e1 proposed the processes of compostable polymer; basically, it is a process of complete decomposition of the plastic (e.g., Cellulose, Hemicellulose) to CO2 and H2O (Mohapatra et al. 2017a). This proposed phenomena after getting lots of criticism are modified to meet the international standards as mentioned below: To meet the above standards, the polymer should have the following characteristics: 1. On composting, it should break down quickly 2. Under the composting, it should quickly biodegrade

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3. Compost should retain utility and value and can support plant life 4. It should be nontoxic and biocompatible. Considering two polymers like biodegradable and compostable plastic, both have their own features such as they need different physicochemical and environmental parameters for their disintegration and the end product of those polymers also make them different from each other. All compostable polymers are by default biodegradable, but not the other way around. Two criteria define a “biopolymer” (or “bioplastic”): (1) raw material source and (2) bio-deterioration. On this basis, the biopolymer can be differentiated into the following types: 1. Type A: biopolymer is biodegradable and made from renewable sources (bio-based). 2. Type B: biopolymer is not biodegradable but made from renewable sources (bio-based). 3. Type C: biopolymer is biodegradable but it’s made from fossil fuels. Type A: Type A biopolymer can be produced (with help of microorganisms, plants, and animals) or chemically synthesized using biological materials like corn, sugar, starch, etc. Type A biopolymer includes: 1. Poly (lactic acid) (PLA) considered as synthetic plastic, which can be polymerized in the lab using renewable raw materials. 2. Some microbial-derived lipid easter-like PHAs are the examples of biopolymers. 3. Biopolymer which naturally occurs, for example, starch and protein. Natural polymers are polymers which are produced in the biosphere by various routes. Starch and PHA are the top-used bio-based biodegradable polymers. Type B: The biopolymers which are produced from renewable sources or biomass and lacks biodegradability are Type B biopolymers. Nonbiodegradable bio-based biopolymers (Type B) include various artificial polymers, synthesized by some renewable base materials, for example, certain polyamides derived from castor oil, other examples: biopolyethylene and propylene, biopoly (vinyl chloride) derived from bioethanol, etc. Type C: Such type of biopolymer are produced from fossil fuel; though some are derived from synthetic aliphatic polyester, they are biodegradable by composting method. Other polymers such as PCL, PBS, and “aliphatic-aromatic”-derived plastic can be deteriorated through microbial enzyme and metabolites. The bioplastic material is defined by European bioplastics; in a broad range, it was categorized based on the origin and degradability nature (Ti et al. 2017) (Fig. 8.1). Thus, sugarcane-derived polymer biopolyethylene is classified as a

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Fig. 8.1 Classification of biopolymers

biopolymer; it is nonbiodegradable but emits a lower amount of greenhouse gases compared to polyethylene; it is nicknamed “green polyethylene.”

8.2

Classification of Biopolymers

Biopolymers are divided on the basis of source of origin point, degradation processes, and its end product. Some of the biopolymers are also derived from fossil fuel, due to nontoxicity and easy biodegradability nature that are considered under biopolymer. On the other hand, the bio-based polymer has its sources produced by microorganisms, plants, and animals. Comparing the degradability of bio-based biopolymer, it has a higher number of nonbiodegradable biopolymers compared to biodegradable bio-based biopolymer (Mohapatra et al. 2016; Ravenstijn 2010). Various categories are mentioned under which the three different types of biopolymers are distinguished (Fig. 8.2). Some are bio-based polymers, PLA being able to be synthesized by both renewable sources and fossil fuel; it is largely produced from renewable sources like starch and sugarcane (Dash et al. 2014; Mohapatra et al. 2016; Mohanty et al. 2014). Nowadays, the volume of bio-based thermoset biopolymers surpasses the volume of bio-based thermoplastic biopolymers. Based on the composition of biopolymers, it can be classified as blends, composites, or laminates (Raquez et al. 2010; Ravenstijn 2010).

8.3

Poly(b-, g-, d-hydroxyalkanoates)

Polyhydroxyalkanoates are having a vast variety of monomeric polymers. Physical properties of PHA lies in a range of Tm between 45 and 182  C, Tg between 56 and 15  C, crystallinity degree between 0 and 80%, and break at an elongation between 5 and 500%. The crystallization rate in a polymer can be controlled (Pati et al. 2020). With reducing molecular weight, the Tm gets reduced; however, Tm remains relatively constant. Elastomers have low crystallinity, an elastomer is PHAs with medium-chain length monomers containing 6–14 C carbon atoms. Poly

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Fig. 8.2 Interrelationship between biopolymers

(3-hydroxybutyrate) (P3HB), poly(4-hydroxybutyrate) (P4HB), and poly (3-hydroxybutyrate-co-hydroxyvalerate) (PHBHV) are examples of short-chain PHAs, whereas P3HB is highly crystalline with Tg of 4  C and a Tm of 180  C (Mohapatra et al. 2015). The polymer PHBHV has been characterized by its lower crystallinity and lowers Tm although its character depends upon its composition of 3-hydroxyvalerate (3 HV) no significant change has been observed in crystallinity and elasticity even on increasing 3-hydroxyvalerate (3 HV) content in polymer (Ravenstijn 2010). Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and poly (3-hydroxybutyrate-co-3- ydroxyoctanoate) are some classic examples of mediumchain length PHAs, medium-chain length PHAs characterized with Tg between 25 and 65  C and Tm between 42 and 65  C (Rodriguez-Contreras 2019). When the composition of 3HH is increased in PHBHHx, it showed a significant change in its properties and the crystallinity of PHBHHx rapidly decreases. This is because 3HB and 3HH have structural difference in two methylene groups in the side chain—cf PHBHV (Mohapatra et al. 2017c). The PHA polymer composed of short-chain length monomers are characterized by little elongation, brittle and easily tear under mechanical constraint, for example, P3HB and PHBHV. On increasing the side-chain of PHA, it will increase the flexibility of polymer, for example, PHBHV is tougher and less brittle compared to P3HB. Likewise, poly(3-hydroxyoctanoate) is an elastomer, and PHAs with longer side-chain has wax-like characteristics (Vroman and Tighzert 2009). P3HB and polypropylene polymers are quite similar in physical and mechanical properties although their chemical structures are quite different, elongation at break

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is an exception here. P4HB has a tensile strength comparably similar to polyethylene, it is ductile, and do not exhibit brittle fracture characteristics. However, P4HB is well comparable with poly(trimethylene carbonate) for mechanical properties. PHBHHx and linear low-density polyethylene (LLDPE) share similar mechanical properties. Polyhydroxyalkanoates (PHAs) are polyesters of β-, γ-, δ-, and ε-hydroxyalkanoic acids (also known as 3-, 4-, 5-, and 6-hydoxyacids or -hydroxycarboxylic acids), PHAs with monomers of chain length in between 3 and 5 carbon atoms are semi-crystalline in nature (Rodriguez-Contreras 2019). PHAs are distinguished based on several factors, mostly by the position of the hydroxyl group concerning the carboxyl group. Moreover, the length of the side chain, a variety of substituents in the side chains, and an additional methyl group on carbon atoms present in between carboxyl group and hydroxyl groups are also considered (Loos 2010). Microorganisms produce a wide range of PHAs based on the chemical structure of the provided carbon substrate, constituents in bacterial PHAs are identified with more than 150 hydroxyalkanoic acids so far (Loos 2010; Steinbüchel and Valentin 1995; Witholt and Kessler 1999). The length of monomers in PHAs affects its properties, most of the PHAs have side-chain monomers in between 3 carbon atoms (3-hydroxypropionate) and 14 carbon atoms (3-hydroxytetradecanoate). Based on the carbon length of monomers, PHAs are classified into short-chain length PHAs (3 to 5 carbon atoms) and medium-chain length PHAs (6 to 14 carbon atoms). On increasing monomer length, it increases the flexibility of polymers. Hence, the PHAs with short-chain length (P3HB) monomers are brittle whereas monomers with medium-chain length are flexible (poly (3-hydroxyoctanoate) (P3HO) is an elastomer). Microorganisms are used for the production of PHAs at a commercial level; in microbes, it is as intercellular carbon and energy storage materials. PHAs are produced naturally by soil bacteria and get degraded on consequent exposure to the same soil bacteria, compost, and marine sediment. PHAs are found as discrete granules inside bacterial cells and can share up to 90% of the bacterial dry cell weight. Biodegradation of PHAs took place when the bacterial cells start growing on their surface and secrete enzymes, as a result, break down the polymer into hydroxy acid and monomeric units. The hydroxy acid obtained from the PHA is utilized by bacteria as a carbon source. The recent advancement has made it possible to produce monomer and polymer synthetically. PHA has opened the scope for the production of polymer from fossilfuel to a renewable source, thus attracts researchers around the globe. PHAs have a vast variety of applications such as consumer packaging, disposable diaper linings, garbage bags, food, and medical products. Moreover, it is well-suited with the traditional production process (Niaounakis 2015). PHAs can have a huge biomedical application, thus it is being extensively studied for their properties and their potential uses in controlled release, to use in the formulation of tablets, surgical sutures, wound dressings, lubricating powders, blood vessels, tissue scaffolds, surgical implants to join tubular body parts, bone fracture fixation plates, and other orthopedic uses (1999, WO9932536 A1, METABOLIX INC.). PHAs have a huge diversity of monomeric composition, and hence polymer can be produced in a range of physical properties. PHAs like P3HB, PHBHV, P3HB4HB, PHBHHx, and several

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others are commercially available and are derived from bacterial fermentations (Alshehrei 2017).

8.4

Applications

Synthetic biodegradable polymers can be used to manufacture numerous products, but its biomedical application dates back to the late 1960s. The first approved biomedical object is bioabsorbable suture. Since then, the use of biopolymers has caused a revolution in biomedical field and manufactured products are not only limited to wound enclosures, body implants, tissue engineering materials but also drug delivery materials for disease treatment and in vivo sensing materials. Biodegradable polymers are suited to be used for medical purposes due to its ability to degrade and convert into nontoxic products and easy elimination from the body without the requirement of alternate specified routes. There are a few other similar terms which can be categorized under the general definition of biodegradable polymers. Although these have been used interchangeably in the literature, there are slight differences in these terms as per their utilization and metabolism inside the body of the living organism. The terms are enlisted below with exact meanings: • Bioresorbable: It refers to the biodegradable polymers that undergo bulk erosion and physiological conditions can be degraded into the products and the products reabsorption takes place within the body itself. • Bioabsorbable: It refers to biodegradable polymers that body fluids can dissolve for their utilization. Furthermore, if the dissolved biopolymer is metabolized and/or eliminated by the body of the consumer as the excretory products, a bioabsorbable polymer can be referred to as bioresorbable. • Bioerodible: It refers to water-insoluble biodegradable polymers. The biopolymers can undergo surface erosion and under physiological conditions, are converted to water-soluble products, employing physical processes like, dissolution and chemical processes, which are resorbed within the body. The process of biodegradation of biopolymers can be mediated by the use of enzymes, the degradation can also occur by hydrolysis (breakdown in the presence of water) and/or possible due to the presence of the other chemical compounds inside the body. Hydrolysis of the ester linkage is the most prevalent method for biodegradation. The other labile bonds and sites which are hydrolytically unstable can also be the target for the degradation. This property of biodegradation of biopolymers is due to its instability that is of utmost importance for its application in biomedical sectors.

8.4.1

PHA Application

Polyhydroxyalkanoates (PHAs) naturally occurring biopolyesters, being produced by several microbes in an enormous variety having a range of properties that

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synthetic polymer didn’t have. Properties like biodegradability, biocompatibility, and nontoxicity are crucial in a material having biomedical applications. Additionally, PHAs can be chemically synthesized or can be produced through fermentation of different combinations of carbohydrates, or adding specific inductors can also do the task. PHAs can be produced in a vast variety having diverse material properties through a conventional production process, therefore having large applicability in different fields like biomedical applications, biofuel, bioplastics, agriculture, and the food industry (Rodriguez-Contreras 2019). Moreover, PHAs are a potential alternative to fossil-fuel-based polymers (Zhang et al. 2009).

8.4.2

PHA as Packaging Materials

The first intended use of PHA was to manufacture packaging materials and bottles. Companies like Wella (Germany), Proctor & Gamble, Biomers, Metabolix, and several other companies used PHA for manufacturing articles such as bottles, utensils, feminine hygiene products, cups, medical-surgical garments, containers, and paper coating (Rodriguez-Contreras 2019; Weiner 1997). PHB fibers with high tensile strength were prepared by stretching the fibers after isothermal crystallization near the glass-transition temperature. However, increasing the isothermal crystallization period decreases the strength of PHB fiber. Extending time (more than 24 h) for isothermal crystallization of PHA fiber significantly increases its tensile strength (Tanaka et al. 2007). High molecular weight drawn fiber has a comparatively lower tensile strength compared to lower molecular weight drawn fiber. Vogel et al. (2007) had successfully improved the crystallization of PHA fiber and disabled the inhibition of secondary crystallization in the fiber. They used reactive extrusion with peroxide for improvement of the crystallization of PHB in a melt spinning process. By this method, they created a very strong fiber with a promising application and overcame the brittleness of PHA.

8.4.3

PHA as Biofuels

Zhang et al. (2009) showed that PHA and PHB can be used as biofuel by producing methyl esters of the PHB and mcl PHA. Transesterification process used to produce methyl esters of PHA and PHB which are 3-hydroxybutyrate methyl ester (3HBME) and mcl 3-hydroxyalkanoate methyl ester (3HAME); comparative study for the combustion heat was done and found that 3HBME and 3HAME had combustion heat of 20 and 30 kJ g 1, respectively, hence comparable with ethanol having 27.1 kJ g 1. Furthermore, they studied different fuels like ethanol, n-propanol, n-butanol, diesel, gasoline, blended fuels of 3HAME, and 3HBME. Ethanol with 10% blend with 3HBME and 3HAME has shown improvement in combustion heat which elevated to 30 and 35 kJ g 1, respectively. However, n-propanol and n-butanol didn’t show any improvement in blendings of 3HAME and 3HBME, rather slightly decreased. In a rough estimation, it was found that it

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would cost US$1200/ton for the PHA-based fuel production (Rodriguez-Contreras 2019; Zhang et al. 2009). Furthermore, esterification of P(3HB-co-3 HV) will produce its methyl esters under optimum conditions (reaction temperature 65  C and time of 60 h) with methanol and 15%(v/v) H2SO4. The highest obtained yield of 3HAME was 74.4% with 97% methyl ester content and its properties were able to meet the required standards of ASTM and Thailand’s fuel standards (Sangkharak et al. 2020).

8.4.4

Biomedical Applicability of PHAs

8.4.4.1 Tissue Engineering The area of tissue engineering is multidisciplinary refers to rejuvenation of injured or infected tissues and organs. Mainly it emphasizes on formation tissues using some biocompatible biomaterials, bioactive molecules, and cells (Waghmare et al. 2018). Tissue engineering can be further classified into hard tissue engineering (bones and cartilage) and soft tissue engineering (vascular and skin graft) (Findrik Balogová et al. 2018). The biomaterial must have the mechanical strength and should serve healthy environment to cell adhesion and proliferation; these features enhance the quality of such biomaterial application in biomedical field (Lizarraga-Valderrama et al. 2019). 8.4.4.2 Hard Tissue Bone Bone tissue engineering deals with the recover and re-formation of new bone tissue by inducing cell growth. To compensate for the required mechanical flexibility, stress, and strain; PHA is optimized by blending it with hydroxyapatite (HA), inorganic substances, hydrogels, and nontoxic, and biocompatible materials. Degli Esposti et al. (2019) developed a scaffold having optimum amount of porosity that helps in bone rejuvenation; the scaffold was developed by mixing PHB with HA particles. Incorporation of HA helps to achieve the osteoinductivity and osteoconductivity in bioactive scaffold polymer. Chernozem et al. (2019) have developed biodegradable scaffolds using CaCO3 with PHB and PHBV and demonstrated comparative characterization. Meischel et al. (2016) have studied the response of bone to PHA composites by implanting it in the femora of growing rats. Cartilage Tissue engineering has opened the scope in area of rejuvenation of infected or ruptured cartilage. However, there are substantial challenges have been observed in cartilage surgery to carry out the function of cartilage as it was working before. To regain the properties of damaged cartilage, there is an utmost need for an alternative of cartilage that matches the properties, durability, and stability of native hard tissue (Rodriguez-Contreras 2019). Several studies have indicated that the PHA could be a potential for the application. A study was done by Ching et al. (2016) to produce a

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polymer mimicking the properties of the naturally occurring cartilage. They experimented with the blends of PHB and poly(3-hydroxyoctanoate) (P3HO) and optimized some of its properties like their structures, stiffness biocompatibility, and degradation rate (Bandekar et al. 2017).

8.4.4.3 Soft Tissue Cardiac Tissue Engineering Cardiac tissue engineering is among the hot topics in the field because of the utmost need due to massive clinical cases. Among the PHA family, mcl PHA is identified with properties essential for cardiac tissue engineering application. Being mediumchain length PHA, they show elastomeric nature and do meet the other needed properties like increased Tg, promising integration of myocardial network, and association of bioactive compounds such as endothelial growth factor enhances cellular viability, adhesion, and growth (Constantinides et al. 2018). P4HB was identified with the potential applications; it can be used for congenital heart defects heart valves and heart grafts. This pliable material is also found helpful in developing absorbable monofilament sutures and among others like ligament repair, hernia, tendon, etc. (Rodriguez-Contreras 2019). Bagdadi et al. (2018) developed multifunctional cardiac patches based on P3HO having properties bordering that of cardiac muscles. It was considered a potential substitute as they intensify cell count, multiplication, adhesion, etc. When it comes to cardiovascular disease, valvular heart disease stands as the third most leading cardiovascular disease. Heart valve tissue engineering (HVTE) is a potential candidate for the treatment (Mohanty et al. 2015). For HVTE, a polymer is essential which can withstand the mechanical environment, guide tissue regeneration, and able to grow with the patient’s heart. Various synthetic biodegradable elastomer which looked for their potential application in HVTE is mentioned in an article by Xue et al. (2017). Wound Healing Wound healing requires a more complex biomaterial which has a significant effect on the regeneration of injured skin, and it is a serious concern of reconstructive medicine (Shishatskaya et al. 2016). For the application in wound healing, several natural components such as collagen, fucoidan, hyaluronic acid, chitosan, and synthetic polymers such as polyurethanes, teflon, and methyl methacrylate have been tested previously as dressing materials (Demir and Cevher 2011). For the application of wound healing, a biomaterial has to meet the characteristics of healthy skin, the biomaterial should have effective antimicrobial property, permits gaseous exchange, promotes a moist wound environment, suitably elastic to fit the wound, and provides the mechanical support (Shishatskaya et al. 2016). Chronic bacterial infection is one of the major factors which inhibit the wound healing process. Thus, research has been conducted on the polymer having antimicrobial properties and antibiotic delivery systems. For example, a wound dressing was designed to have anti-biofilm protein-releasing properties; they embedded Dispersin B (DB) in an asymmetrical PHA substrate (Marcano et al. 2017).

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8.4.4.4 PHAs for Organ Tissues Among the PHA family, PHBVHHx is found to have a significant property to support the growth of stem cells. In some studies, PHBVHHx has been used to prepare the supportive scaffolds (three-dimensional) for organ tissue. In several studies, the film and scaffolds were loaded with the stem cells of the human umbilical cord (UC-MSCs) to regenerate the injured liver tissues. In a study, the liver-injured mouse was transplanted with the PHA scaffold biopolymer, and it has shown a positive response in the recovery of the injured liver (Su et al. 2014). Tashjian et al. (n.d.) have found PHA promising for healing tendons. They produced bioresorbable scaffolds to strengthen the suture-tendon interface in the rotator cuff repairs. In a study, a blend of PLA and PHB was developed which was aimed to replace the urethra tubule (Findrik Balogová et al. 2018). 8.4.4.5 PHA for Drug Delivery Systems The reason for PHA being a potential polymer for drug delivery is because of its biodegradability under different environments. PHA is hydrolyzed into oligomer and monomer by several microbes secreting PHA-hydrolyzing enzymes extracellularly and utilizing these oligomers and monomers as nutrition (Rodríguez-Contreras et al. 2012; Tokiwa and Calabia 2004). Bacterial depolymerase and hydrolytic mechanisms take more than 52 weeks to degrade PHA (Misra et al. 2006). Besides, several comparative studies compare the degradability of PHA with synthetic and semisynthetic polymers. A comparative study was done by Gil-Castell et al. (2019) to know the durability of different scaffolds which were PHB, polycaprolactone (PCL), PLGA, and polydioxanone (PDO). They found PCL and PHB are more durable than PLGA and PDO; Hence, PDO and PLGA will be suitable for short-term applications and PHB and PCL are the better options for long-term application. However, several factors govern the biodegradability of PHA; factors like biopolymer composition, molecular mass, stereoregularity, crystallinity, and environmental conditions like temperature, pH, moisture, and nutrition supply plays a major role. Studies have shown that increasing the crystallinity of polymer decreases the biodegradability rate while biopolymers with lower molecular mass biodegrade quickly (Errico et al. 2009). Fabrication of PHA can be done in the desired copolymer composition and molecular mass, which makes it a potential biopolymer for the application of drug delivery. Moreover, several properties like its less toxicity, substantial impact on drug bioavailability, and improved encapsulation have already been demonstrated (Li and Loh 2017). Masood et al. (2016) discussed the inferences of the encapsulated anticancer agent within different nanoparticles (PHA, cyclodextrin, and poly lactic-co-glycolic acid) are employed to accurately target the tumor sites. Michalak et al. (2017) reviewed the scientific advancement in the production of PHAs functionalized for multifunctional PHA nanoparticles, micelles, PHA for drug delivery, and conjugates of PHA-drug and PHA-proteins. Li and Loh outlined the advancement in therapeutic delivery carriers through the implication of PHA-based nano-vehicles (Li and Loh 2017). Scientists all around the world have studied different structures of the

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PHA-based delivery system. For example, a system was developed by Lee et al. (2004), by which a metal stent was coated (through electro spun onto a metal stent) with drug-containing PHA fiber to make a biocompatible stent. For drug delivery, PHA has been used as a matrix to release drug formulation at the desired rate; based on the demand the scaffold casted with antimicrobial and anti-inflammatory agents to nullify the virulence factors. Manero’s group studied extensively on PHAs scaffold having diversified cell and tissue development compound coatings (Rodríguez-Contreras et al. 2017, 2019).

8.5

Conclusion

Polyhydroxyalkanoates are renowned bioplastics and the beauty of the polymer is it can be molded in a diversified application based on the monomeric composition, physical and mechanical properties. Basically, these are fatty esters accumulated in the microbial cytosol that serve carbon neutrality. The major challenge of PHAs is the production cost; hence, extensive research is highly essential to reduce the production cost. Moreover, PHAs can be the best biomaterial for a biomedical applications that can compensate for the production cost. Due to the promising characteristics of PHA and its composites, nowadays it is gaining more market in biomedical and research into this area.

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Microbial Pigments and Their Application Selvaraju Vishnupriya, Sundaresan Bhavaniramya, Dharmar Baskaran, and Arulselvam Karthiayani

Abstract

Microbial pigments are versatile with potential application in food, pharmaceutical, cosmetics, and textile industries as a promising natural source of colorant. The diverse group of microorganisms under stress produces pigments that are biodegradable, non-carcinogenic with evident biological and functional properties. Many new pigment producing organisms are now the most sustainable way to overcome the use of artificial pigments that are harmful. This review mainly focuses on the application of microbial pigments in various industries including the benefits and challenges of using natural pigments with the focus on meeting safety and demand. Keywords

Microbial pigments · Industrial application · Food industry · Textile industry

9.1

Introduction

Microbial pigments production is a promising field of research implying demand in various industrial application. Biodegradable or bio-based pigments create awareness for advantages such as eco-friendly, has low toxicity compared to synthetic materials. Bio-colorants are in global attraction estimating 45% of total demand in S. Vishnupriya (*) · S. Bhavaniramya · A. Karthiayani College of Food and Dairy Technology, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India D. Baskaran Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_9

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various sectors such as pharmaceutical, cosmetics, and food. Microorganisms are in interest to isolate pigments and develop organic food colorants. Synthetic colors have developed several health deformation and have artificial impact on the outcoming product or design. Hence, moving forward towards natural colors and pigments from microbial sources has showed a wide array of opportunity in various fields. Natural pigments are used in food industry as additives, colorants, and also in other industries such as textiles, cosmetics, and pharmaceuticals for various purpose. Microorganisms and plant sources-based pigments and colorants are studied to replace the carcinogenic and chemically produced colorants. Biopigments play a vital role in global market providing demand for organic pigments, dyes. Furthermore, covering the food industry the focus turns out to be for thickening agents such as xanthan and gellan, flavor producers, essential amino acids, biopigments, viz., carotenoids, flavonoids, melanins, quinones, etc. Microorganisms, especially Monascus, Rhodotorula, Bacillus, and Achromobacter are capable of producing a large number of biopigments and they are fondly noticed in soil, coast, or deep sea. These pigments also play a supportive part for maintaining the biological activity in food supplements, probiotics, etc. Several bacterial pigments are used for developing colorants such as zeaxanthin (yellow color) extracted from Flavobacterium sp. and Paracoccus sp., carotenoids (yellow) isolated from Streptomyces sp. Excellent advantages of these pigments include non-toxic, biodegradable, non-carcinogenic, eco-friendly. The reach for natural, sustainable food colors from organic pigments is increasing and the perspective to consume natural products replicates in producing innovative ideas. Microbial pigments through natural fermentation serve a low cost of production and microbes produce various substances such as carotenoids and flavins and they are further utilized in food ingredients. Advanced techniques in biotechnology and metabolic science are providing active production of valuable microbes of interest. Further understanding the biosynthetic pathway of isolated pigments production helps in recognizing conditions and strategies of involved microorganisms suitable for developing the low cost pigment extraction methods. Emerging technology alongside application of natural pigments is effectively utilized using nanotechnology as nano colorants and application in various industry such as the food industry and textile industry are in focus to be developed from microbial pigments. Environmental pollution such as aquatic animal contamination, soil and groundwater remarks are a serious issue with the use of synthetic pigments and therefore more interest on natural products are in high demand.

9.2

Source and Production of Microbial Pigment

The production of pigments by microbes from various sources ranging from soil, marine to agro-industrial and food waste has a prospective demand as they come with the future dimension of having eco-friendly products. These pigments are produced by fermentation of organic waste such as agro-industrial waste otherwise from the nature especially marine source or soil as shown in Fig. 9.1.

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Fig. 9.1 Source of Pigment Producing Organism

9.2.1

Agri-Industrial Waste

Waste generated during the processing of agricultural produce or production is called agro-industrial waste. These waste mostly go underutilized and cause environmental pollution despite the fact it is highly nutritious based on the source (Sadh et al. 2018). However, the waste can be converted to wealth by treating the waste such as seeds, peel, and pulp. Among which microbial pigments are the important value-added product replacing the harmful artificial synthetic colorants and dyes. A wide range of microbes such as bacteria, fungi, yeast, and algae produce a compendium of pigments opening opportunities for various industries (Heer and Sharma 2017). Pigments such as carotenoids have various advantages such as antioxidant, antibacterial properties. Abdelhafez et al. (2016) used Serratia marcescens for the pigment production using agri waste rice bran, sugarcane bagasse, and molasses as substrate. When compared to the other waste residues, molasses served as the best medium with the maximum production of β-carotene (1.1 mg/L) in a rotary shaker at rpm of 150 for 2 days incubated under dark condition with the temperature of 30
C. Similarly, using Serratia marcescens Xd-1prodigiosin was produced by solid-state fermentation of bagasse. Using ultrasonic assisted reflux extraction of higher amount of prodigiosin (40.86 g kg1 dry solid) was obtained with the addition of glycerol and soy peptone (Xia et al. 2016).

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From the cannery industry solid waste was utilized for the pigment production, pineapple waste was used for violet pigment at low cost in large scale for industrial application. C. violaceumUTM5 were used to produce violet pigment at optimum condition of pH 7, temperature 25–30
C while using liquid pineapple waste. When compared to the pigment production low amount of violet biocolor was produced using nutrient broth (239  3 mg L1) while pineapple waste produced 285  5 mg L1 (Aruldass et al. 2015). The same author using Chryseobacterium artocarpi CECT 8497 replacing nutrient broth with pineapple waste as nutrient source supplemented with nitrogen and phosphate source for the production of yellowish-orange pigment which is utilized in the soap making process. The optimum condition for the pigment production was carried out in 50 L bioreactor at 30
C for 24 h at 200 rpm (Aruldass et al. 2016). One of the most widely available wastes is bakery waste (BW) to utilize them effectively Haque et al. (2016) used Monascus purpureus for pigment production using the BW hydrolysates as nutrient-rich media in solid-state fermentation and submerged fermentation. The highest pigment (about 24 AU/g glucose) obtained under the hydrolysis condition of temperature was 50 and 55
C along with glucose concentration of 5 g L1 in submerged fermentation and also reported to have no adverse effect when used as food colorant. Similarly, Sehrawat et al. (2017) using Monascus purpureus produced pigments using different wastes such as sweet potato and potato peel powder, kinnow peel, also rice and wheat bran, as carbon source to compare the maximum yield. After 15 days of fermentation, it was observed to have a maximum amount (3.3 CVU/g) of biopigment was obtained when sweet potato peel powder was used. Followed by this orange waste was utilized by fungal strain Monascus purpureus ATCC 16365 and Penicillium purpurogenum CBS 113139 for the pigment production using submerged fermentation, solid-state fermentation, and semi-solid-state fermentation for the maximum yield. In comparison Monascus purpureus under solid-state fermentation obtained the maximum production of 9 AU/g with high total phenolic content (123.7 mg EAE mL1) of dry fermented substrate (Kantifedaki et al. 2018). With sugarcane bagasse (SCB) hydrolysate as a carbon source Hilares et al. (2018) red biocolor was produced using Monascus ruber Tieghem IOC 2225. Monascus ruber grows well on glucose-based medium however using SCB the yield was 2.5-fold higher under dark conditions after 288 h of fermentation. Different incidences of light were used to compare the yield of red pigment under white light (12.13 AU 490 nm), dark condition (18.71 AU 490 nm) while the biomass under dark condition was (5.78 g L1). In addition to this, de Paula Bonadio et al. (2018) using the sugarcane juice produced carotenoids with Rhodotorula rubra L02. Among the 16 different combinations, it was obtained that the complex sugarcane juice and synthetic media with maltose and sucrose showed best results of about carotenoid production 0.0135 mg/L, 0.0277 mg/L, and 0.029 mg/L, respectively. The coffee waste was utilized for the biopigment using Rhodotorula mucilaginosa CCMA 0156 (Collection of Microorganisms of Agricultural Microbiology) with high antimicrobial and antioxidant activity. The maximum yield of pigment using pulp extract was 16.36  0073 mg L1 followed by husk with the yield of

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21.35  0067 mg L1. The antioxidant activity of the pigment from pulp extract (98.26% Protection β-carotene) was comparatively higher than the synthetic antioxidants (BHT—85.92% Protection β-carotene) (Moreira et al. 2018). Similarly, using the corn cob Embaby et al. (2018) using Monascus purpureus strain ATCC 16436 through co-solid-state fermentation 10 days at 30
C orange and red pigments are produced. In another study, Patthawaro et al. (2019) using Rhodopseudomonas faecalis PA2 with different agro-industrial wastes such as cassava meal, soybean meal, and coconut meal for the carotenoid production. As a result, under favorable condition lycopene,1,2-dihydro-3,4-dedihydrolycopene, cis-1,2-dihydrolycopene, 1,2-dihydrolycopene were produced when using soybean meal. These photosynthetic bacteria produced the major carotenoid with only 50% soybean meal in the medium while the pigment production was very low in cassava and coconut meal. The lycopene has good antioxidant effects and bioactive functional properties due to the presence of polyunsaturated hydrocarbons. Followed by the above study Saejung and Puensungnern (2020) also studied using Rhodopseudomonas faecalis PA2 for producing carotenoid replacing chemical medium with molasses-based medium while reducing the cost. This photosynthetic bacterium is one among the most preferred commercial sources of carotenoid production that can be used in fortification of food. Blakeslea trispora (+) MTCC 884 was used along with orange, papaya peels, and carrot waste under solid-state fermentation resulted yield of 0.127 mg/mL of β-carotene a red-orange pigment. For the confirmation of color mass spectroscopy and LCMS was used whose m/z value was 537.608 and peak value Rt 13.37. Apart from this, the extracted β-carotene had a scavenging property for about 90 days. At pH range of 6.2–6.5 with temperature between 25 and 32
C under fermentation of 60–96 h the optimum β-carotene production was obtained (Kaur et al. 2019). Similarly, using mung bean husk, onion peels, pea pods, and potato skin Sharma and Ghoshal (2020) produced biopigment from Rhodotorula mucilaginosa MTCC1403 in the bioreactor. They obtained the optimum carotenoid production (717.35 μg/g of β-carotene) at temperature of 25.8
C with the pH 6.1 and agitation 119.6 rpm using flask while under aerobic fermentation the yield was higher 819.23 μg/g which shows carotenogenesis vary with aeration under same condition. Under submerged fermentation using Monascus ruber different pigments were produced by varying the pH of maltose syrup residue when used as substrate. At pH 2.0 and 2.5 (highly acidic) yellow pigments were produced, orange pigment when pH was 3.0 and 3.5 and red with pH 4.0 and above while potentially utilizing the waste from the syrup processing industry (De Oliveira et al. 2019).

9.2.2

Marine Source

Compared to soil or terrestrial organisms recently marine-sourced (animals, sponges, sea urchins, marine crustaceans) are gaining a lot of importance with the potential to

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produce different pigments. These pigments have various features such as scalability, non-toxicity, eco-friendly, and easy gene manipulation (Bălașa et al. 2020). From the coral reef of the Reunion Island, isolated a Penicillium fungi Talaromyces albobiverticillius 30,548 for pigment production. During the study, they identified 12 compounds among which only 4 (N-glutaryl rubropunctatin, N-threonine-monascorubramine, 6-[(Z)-2-Carboxyvinyl]-NGABA-PP-V, and PP-O) were Monascus-like red pigment with no mycotoxin. Under submerged fermentation for 8 days at temperature 24
C with the agitation speed of 150 rpm. The red azaphilone biocolorant was confirmed using NMR analysis with the cis configuration at the C10-C11 double bond (Venkatachalam et al. 2018). From the South Yellow Sea in China the researchers isolated Rhodotorula sp. RY1801 under fermentation with pH 5.0 and temperature of 28
C for the production of carotenoids of about 589 to 987 μg/L along with glucose, yeast extract, carbon, and nitrogen sources (Zhao et al. 2019). Two organisms isolated from the Island of La Reunion for pigment production were Talaromyces spp. and Trichoderma atroviride. Using submerged culture fermentation both the extracellular and intracellular pigment were extracted using pressurized liquid extraction. N-threonine rubropunctamine and Monascus-like azaphilone were the pigments produced and characterized as non-toxic and non-mycotoxigenic (Lebeau et al. 2017). In addition, many novel pigment producing organisms were isolated and identified from marine environments. Similarly, from the same region various samples such as seawater sediments, living and rubble corals and many others were taken. Among which 31 different species were identified with major genera being Penicillium, Aspergillus producing pigments from dark shades to light shade when using PDA medium with wide application in pharmaceutical, fertilizer, and agro industries (Fouillaud et al. 2017). Marine organisms are the major producers of anthraquinones, Stemphylium, Fusarium, Eurotium, Microsphaeropsis, and Paecilomyces play a major role in the production. Apart from these Xylaria sp., a filamentous fungus produced a new compound called xylanthraquinone, Aspergillus glaucus produced aspergiolide A, P. chrysogenum produced skyrim from the Salt Lake, Fusarium sp. produced anhydrofusarubin, Nigrospora sp. produced 4-deoxy bostrycin and various other marine-derived fungus has pigment producing property with good antioxidant activity (Fouillaud et al. 2016). In another study in Italy, sea sponge samples were taken from which Raspaciona aculeata and Dictyonella marsilii were isolated for the carotenoid production. A typical carotenoid called renieratene, a brown colored pigment, was found in both the species but predominantly in R. aculeata 2570 ppm while D. marsilii had 277.1 ppm of carotenoid (Salvo et al. 2017). In the marine ecosystem, astaxanthin is the most common produced by photosynthetic organisms. From the marine water of pacific coast which in the middle Japan the researchers isolated Brevundimonas genus a novel strain N-5 producing astaxanthin an important carotenoid pigment on the global market. Under good aerated conditions, these orange-pigmented bacteria (N-5) produces optically pure carotenoid of 364.6 μg g1 dry cells while the total amount was 601.2 μg g1 dry cells. Apart from this other carotenoid such as 2-hydroxy astaxanthin, and

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2,20 -dihydroxy astaxanthin were also identified using their retention time in the HPLC (Asker 2017). The same author studied the keto carotenoids such as astaxanthin, adonixanthin, and their hydroxylated derivatives from fresh and marine water, among them Brevundimonas and Erythrobacter were the highest producers. Different strain from freshwater were isolated FrW-Asx-5, FrW-Asx16 produced astaxanthin content of 46% and 16.6% and the other derivatives 2,20 dihydroxy astaxanthin, 2-hydroxy-astaxanthin are also produced (Asker 2018). For surface seawater, 88 bacterial strains belonging to Bacilli, Actinobacteria, Flavobacteriia, α-Proteobacteria, and γ-Proteobacteria classes were isolated for the production of different carotenoids such as canthaxanthin astaxanthin, lutein, and zeaxanthin. Among the several pigment producing strains Flavobacterium produced 93.1% zeaxanthin of total carotenoids. They have studied both the upper and deep seawater to isolate the pigment producing bacteria; the upper layer had a greater number of bacteria by protecting itself from UV light using pigments (Asker et al. 2018). Similar to the above study recently, from the scallop pigment producing Brevundimonas was isolated. 2,20 -dihydroxy-astaxanthin (82% of total carotenoid) was the major carotenoid obtained from the marine bivalve exhibiting excellent antioxidative activity due to the presence of the extra hydroxyl group. Compared to the other source of astaxanthin producing bacteria, B. scallop produced 1303.62  61.06 μg/g dry cells. Low temperature and salinity with high carbon source are the optimum for the carotenoid production (Liu et al. 2020).

9.2.3

Soil

Pigments produced from soil organisms have a huge scope of application in various fields such as cosmetics, medical, food, and textile industry as colorant. Biopigments owing to its non-toxicity have the other advantage of being simple and quick to grow independent of the climatic condition. Various soil samples from different places were taken for the isolation of pigment producing microorganism Penicillium spp. from the Brazilian cerrado soil, P. sclerotiorum isolated from National Park of Serra Do Cipo produced pencolide, sclerotiorin, and isochromophilone followed by P. oxalicum for the production of hydroxyanthraquinone by a Czech company from the soil fungal strains (Akilandeswari and Pradeep 2016). Bacillus weihenstephanensis was isolated from one of the coldest regions of Northeastern Poland (National park and Farm) for the production of melanin-like pigment with good solubility. During this microbial melanogenesis process, laccase activity contributed to the pigment production, six B. weihenstephanensis were melanin-positive isolates among the various samples. However, the B. weihenstephanensis from the farm soil exhibited higher pigment production than the park soil sample due to differences in the soil fertilization (Drewnowska et al. 2015). Biopigments from Streptomyces fradiae were used in various industries for its hemolytic and antioxidant properties. The organism was isolated from the soil of Sundarbans forest and was tested to replace the synthetic colorants with the eco-friendly biopigments in the textile industry (Chakraborty et al. 2015). Similarly,

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Yasmeen et al. (2017) isolated black pigment producing Streptomyces species from the mangrove forest for its potential antimicrobial activities from Andhra Pradesh. Forest soil from South Korea was used for isolating yellow and flexirubin-type pigment producing Dyadobacter genus belonging to family Cytophagaceae (Dahal and Kim 2018) and soil bacterium Flavobacterium humisp also produced flexirubintype pigment Kim et al. (2019), while Park et al. (2020) isolated Chryseobacterium from mountain soil from Korea reporting the same. He also isolated Deinococcus species for deinoxanthin production; this major carotenoid produced by D. radiodurans was studied by Kumar et al. (2015) for its antitumor property. Lebeau et al. (2017) analyzed and isolated Penicillium purpurogenum rubisclerotium, Fusarium oxysporum to replace the synthetic red color, the production of water-soluble biopigment was increased either by increasing the fungus growth or the intracellular pigment. The major fungus species isolated from soil are Penicillium and Aspergillus that is P. citrinum, P.aurantiogriseum from forest soil P. corylophilum, P. fellutanum, A. parasiticus were isolated from tea plantation while A. flavus, A. glaucus were from Rhododendron soil, (Pandey et al. 2018a). From the Indian Himalayan region, orange color pigment producing Penicillium sp. was isolated which was produced under stress due to weather conditions. The pigment was insoluble and found to be derivative of carotenoid (Pandey et al. 2018b). Apart from this Penicillium striatisporum isolated from Australia and Penicillium purpurogenum produced yellow pigments, while Fusarium verticillioides produced naphthoquinone pigment isolated from Thailand soil (Kalra et al. 2020). The mesophilic bacteria Salinococcus roseus produced temperature stable orange pigment due to the presence of double bond that are mostly organic compounds when obtained from soil. Hence, it can have various application in textile and food industry (Usman et al. 2018). Fariq et al. (2019) from Punjab, Khewra Salt Range, soil samples were collected for the isolation of halophilic microbes such as A. elongatus, H. aquamarina, and S. sesuvii for the production of bacterioruberin carotenoids. Resembling this study, soil samples from various places of Gujarat were taken for the pigment producing bacteria among wide group Staphylococcus xylosus was identified and also found under stress or harsh condition pigments produced were used in the cosmetic industry (Choksi et al. 2020). Chaetomium cupreum was isolated from litter soil for the red colored pigment collected from Karnataka under submerged fermentation and evaluated the extract for its antioxidant activity and application in pharmaceutical (Wani et al. 2020). A blue green phenazine pigment from Pseudomonas aeruginosa produce pyocyanin isolated from the soil dumped with agri waste in Dharwad, Karnataka. This pigment has commercial value as it used to resolve multiple environmental and agricultural problems (DeBritto et al. 2020)

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201

Application of Microbial Pigments in Various Industries

Microbial pigments are the metabolic secretion of microbes that has a wide application in food, textile, and cosmetics industries owing to its beneficial properties. Being eco-friendly these pigments are non-carcinogenic as well as non-toxic in nature acting as a potential replacer for synthetic pigments. These pigments have remarkable therapeutic values with challenges and opportunities in the pharmaceutical industry. Further discussion on extraction methods of the diverse pigments, its scalability from pilot model to industries and to improve the commercial value of any pigments are with a possible road map of pigment extraction to possible applications in various industries as shown in Fig. 9.2 discussed.

9.4

Role of Microbial Pigments in Food Industry

In the food industry, color plays integral part between the acceptance and taste of the food. The more the attractive color of the food the more it has the influence on the selection of food. Synthetic color are generally anticipated for the application in food industry as well in the cosmetic industry as it is cheap and stable meanwhile associated with the harmful effects forced a transition shift from artificial colors. The growing consumer demand for healthy product and increasing awareness on food safety made the bicoloration best alternative to take over the synthetic pigment (Tuli et al. 2015). In spite of having natural pigments from various sources, microbial sources are safe to use without any curtailment in the production due to weather, raw material, cost, and other technical challenges (Panesar et al. 2015). The apparent reason for eschewing artificial colors and additives in foods is due to its allergic effect and hyperactivity in children in the long run when consumed continuously. Furthermore, certain artificial colors are also banned due to the deleterious teratogenic outcome concurrently; most of the biopigments are anticancer, antifungal with antioxidant capacity (Oplatowska-Stachowiak and Elliott 2017). The essentially increasing need for natural and healthy products in the food industry paves way for researchers to discover new sources of pigment producing organisms and their pigment properties with its varied application to seek demand from the food industry. To improve the color nutrition pigments like carotenoid, riboflavin is used in beverages, snacks, desserts, and also in fresh fruits and meats. Additionally, Fusarium sporotrichioides, a fungus and Erwinia uredovora were used in food preparation to increase their visual quality added to the food to increase its appeal (Narsing Rao et al. 2017). In addition to this, FDA has approved pigments as follows: carotene, astaxanthin, and riboflavin from Monascus, Xanthophyllomyces, and Eremothecium in such a way uplifting the natural source of pigments while meeting the legislation (Numan et al. 2018).

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Fig. 9.2 Road map of pigment extraction to possible applications in various industries

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Microbial Pigments as Food Color

Considering the organoleptic property, visual impression is the major parameter when it comes to food choices also quality of the food is decided by the color and hence to intensify the color of the meat and egg combination of carotenoid (lutein, astaxanthin, and β-carotene) has been used to feed poultry for the good quality product (Ram et al. 2020a, b). While Venil et al. (2015) used violet biocolor powder in jelly and yogurt produced by Chromobacterium violaceum UTM 5 as shown in Table 9.1. The extracted pigment was concentrated using a spray dryer with Gum Arabic as encapsulating material for good stability. When analyzing the product quality, the product was stable with no color change or microbial contamination for 1 month in yogurt and jelly. Similarly for the candy preparation carotenoid extracted from Rhodotorula mucilaginosa was used when fermented using malt and yeast medium with date syrup as carbon and nitrogen source for the maximum production (Elsanhoty et al. 2017). According to Galaup et al. (2015), coloring of cheese rind is a difficult process but with the presence of Brevibacterium linens and Brevibacterium aurantiacum on the surface of vieux-pan French cheese (soft red ripened cheese) resulted in the surface color. Similarly, another Protected Designation of Origin cheese from France called Fourme de Montbrison was investigated for the presence of carotenoid produced by Brevibacterium linens. They found chlorobactene and Agelaxanthin A with nine other carotenoids which are responsible for the rind color (Giuffrida et al. 2020). For the red color of the cheese, meat, wine biocolor produced from Monascus was used and they are also used to color red koji dates using rice as a fermentation medium (Cardoso et al. 2017). Among the microbial pigments Monascus has an important place to exploit as a colorant, additive in the food industry. The major contributors of pigment production are Monascus M3428 Monascus ruber CCT 3802, Monascus purpureus N11S while showing strong antimicrobial activity. With the hydrolytic enzymes, changes can be made in the morphology of Monascus for the pigment production (Kim and Ku 2018). Nitrite is used in the meat industry for the color development but due to its carcinogen production an attempt to replace it with Monascus pigment was studied. For the minced meat, Monascus ruber MJ-1 pigment was added and after 4 days of fermentation proteolysis and lipid oxidation were analyzed. The result showed inhibition of lipase activity followed by reduction in the thiobarbituric acid reactive value and proved to be effective for the fermented meat application (Yu et al. 2015). Followed by meat to deepen the fish color carotenoid was used along with the feed, in particular to improve the color of the red snapper fish (Pereira da Costa and Campos Miranda-Filho 2019). Red color pigment from Meiothermus sp. isolated from the water sample can be used as it showed strong antioxidant activity (Mukherjee et al. 2017). Similarly, blue green algae or the cyanobacteria produced red pigment from Arthrospira platensis, it is generally a protein complex which could also be used to replace synthetic color as it is produced using rice husk as substrate medium (Taufiqurrahmi et al. 2017).

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Table 9.1 Different colors of pigments and its application Red Pigment Microbes

Fungi

Bacteria

Algae

Name of micro organism

Pigment name

Application / Uses

Monascus sp.

Monascorubramine, Rubropunctamine

Antioxidant

Penicilliumoxalicum

Anthraquinone

Anticancer effect in food and pharmaceuticals

Penicilliumpurpurogenum

Mitorubrinol

Dyeing of cotton fabrics

Monascuspurpureus

Azaphilones, Rubropunctamine

Dyeing of cotton fabrics , Wool

Talaromycesaustralis

Carotenoids, 2, 4-Di-tertbutylphenol

Dyeing of cotton fabrics and Wool

Talaromycesverruculosus

Polyketide

Cotton Fabric

Scytalidiumcuboideum

Quinones

Bleached cotton, Spunpolyamide, Spun polyester, polyacrylic, wool

Fusariumsporotrichioides

Lycopene

Antioxidant

HaematococcusPluvialis

Astaxanthin

Antioxidant

Talaromycesatroroseus

Azaphilones

Antioxidant, Anticancer, Antioxidant

Pseudoalteromonasdenitrificans

Cycloprodigiosin

Antiplasmodial, Anticancer

FusariumSporotrichioides, Blakesleatrispora

Lycopene

Antioxidant, Anticancer

Cordycepsunilateralis

Naphtoquinone

Anticancer, Antibacterial, Trypanocidal

Epicoccum nigrum

Carotenoid

Antioxidants, food coloration, Inhibits HIV-1 replication

Lecanicilliumaphanocladii

Oosporein

Antifungal activity

Monascusruber

Azaphilone

Foods

Rugamonasrubra, Streptoverticilliumrubrireticuli, Vibrio, gaogenes, Alteromonasrubra, Serratiamarcescens, Serratiarubidaea

Prodigiosin

Anticancer, immunosuppressant, antifungal, algicidal; dyeing (textile, candles, paper, ink)

α-Proteobacteria

Heptylprodigiosin

Antiplasmodial

Pseudoalteromonasrubra

Prodigiosin

Anticancer, DNA Cleavage, Immunosuppressant

Streptomyces echinoruber

Rubrolone

Antimicrobial

Streptomyces sp.

Undecylprodigiosin

Antibacterial, Antioxidative, UVprotective, Anticancer

Proteobacteria

Heptylprodigiosin

Antiplasmodial

Dunaliellasalina

β-carotene

Porphyridiumcruentum and many other microalgae and cyanobacteria

Phycoerythrin

Antioxidant, Antitumor activity, Immunoregulatory

Orange Pigment Monascus sp.

Monascorubrin, Rubropuntatin, Canthaxanthin

Antibacterial activity, Anticancer activity

Penicilliumpurpurogenum

Mitorubrin

Food, pharmaceuticals and cosmetics

Penicilliumsclerotiorum

PencolideSclerotiorin

Antibacterial activity, Antifungal activity

Neurosporaintermedia

β-Carotene

Various industrial and pharmaceuticals applications

Neurosporasitophila

Neurosporaxanthin

Antioxidants

Epicoccumnigrum (CML2971

Orevactaene

Antioxidant

Fungi

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Table 9.1 (continued) Bacteria Algae

Bradyrhizobium spp. , Lactobacillus pluvalis

Canthaxanthin

Anticancer, Antioxidant

Dunaliellasalina Microalgae

β- carotene

Anticancer, Antioxidant suppression of cholesterol synthesis

Yellow Pigment Aspergillussclerotiorum

Neoaspergillic acid

Antibacterial activity

Aspergillusversicolor

Asperversin

Antifungal activity

Fusariumverticillioides

Naphthoquinone

Antibacterial activity

Monascus sp.

Monascin, Ankaflavin

Food colorant, Pharmaceuticals

Penicilliumherquei

Atronenetin

Food additive, Antioxidant

Penicilliumpurpurogenum

Purpurogenone

Dyeing of cotton fabrics

Trichodermaviride

Viridin

Textile dyeing

Trichodermavirens

Viridol, Virone

Antifungal activity

Ashbyagossypi

Riboflavin

Xanthomonasoryzae

Xanthomonadin

protection against photo damage

Candida famata

Riboflavin

Baby foods, breakfast cereals, fruit drinks

Eurotium spp.

Isoquinoline

Antifungal activity

Penicilliumflavigenum

Anthraquinones

Antioxidants

Scytalidiumganodermophthorum

Quinones

Textile

Fungi

Bacteria

Algae

Monascuspilosus

Citrinin

Thermomyces sp.

Anthraquinones

Cotton, Silk, Wool

Trichoderma sp.

Anthraquinones

Cotton, Silk, Cotton Silk

Aspergillus sp. AN01

Asperyellone

Cotton, Silk, Synthetic and wool fabrics

Penicilliummurcianum

Carotenoids

Wool

Cytophaga/Flexibacteria AM13,1 Strain

Tryptanthrin

Antioxidant, Anticancer

Staphylococcus aureus

Zeaxanthin

Photoprotectant, Antioxidant

Kocuria sp.

Carotenoids

Anti-cancer activity

Pseudomonas sp.

Phenazine

Biological control

Bacillus sp.

Riboflavin

Nutritional supplement

Bacillus subtilis

Riboflavin

Nutritional supplement

Flavobacterium sp., Paracoccus sp..

Zeaxanthin

Photo protectant, antioxidant

Lutein

Antioxidant

Chlorella and others, Microalgae

Brown Pigment Aspergillus sp.

Quinones

Cotton, Silk, Silk cotton

Acrostalagmus (NRC 90)

Quinones

Wool

Bisporomyces sp.

Quinones (Deep Brown)

Wool

Penicilliumchrysogenum (NRC 74) , Penicilliumitalicum (NRC E11), Penicilliumregulosum (NRC 50)

Quinones

Wool

Black Pigment Fungi

Bacteria Yeast

Aspergillus niger

Aspergillin

Antimicrobial activity

Curvularialunata

Anthraquinones

Silk, Wool

Pseudomonas guinea

Melanin

Antioxidant activity

Saccharomyces, Neoformans

Melanin

Antimicrobial, Antibiofilm and antioxidant

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Table 9.1 (continued) Green Pigment

Bacteria

Chlorociboriaaeruginosa

Quinones

Bleached cotton, Spun polyamide,

Pseudomonas Spp.

Phycocyanin

Cytotoxicity, Neutrophil apoptosis, Ciliarydysmotility, Proinflammatory

Bacillus cereus

Azaphenanthrene

Anticancer, Antibacterial, Textile dyeing

Blue Pigment Fungi

Bacteria

Algae

9.4.2

Aspergillusniger

Aspergillin

Antimicrobial activity

Pseudomonas Spp.

Phycocyanin

Cytotoxicity, Neutrophil apoptosis, Ciliarydysmotility, Proinflammatory

Corynebacteriuminsidiosum

Indigoidine

Protection from oxidative stress, Antioxidant, Antimicrobial

Erwiniachrysanthemi

Indigoidine

Protection from oxidative stress, Antioxidant, Antimicrobial

Arthrospira sp. (formerly Spirulina sp.) and many other microalgae and cyanobacteria

Phycocyanin

Antioxidant, Antitumor, Immunoregulatory

Biopigments as Food Additive with its Antioxidant Property

Rhodotorula mucilaginosa AY-01 isolated from the soil collected from Incheon, South Korea was analyzed for the presence of carotenoid as glucose. These glucosidal carotenoids red pigment exhibited strong antibacterial and antioxidant activity (Yoo et al. 2016). Another important pigment from the carotenoid family is zeaxanthin, the International public health organization WHO and FAO recommends about 0–2 mg kg1 zeaxanthin of the body weight (Ram et al. 2020a). From the freshwater zeaxanthin producing Arthrobacter gandavensis was isolated for its application in the food and feed industry as additive for its antioxidant property and also in the cosmetic industry due to its prophylactic activity acts as antiaging agent (Ram et al. 2020b). A novel biopigment isorenieratene, an aromatic carotenoid was isolated from Rhodococcus sp. B7740 for the food application. The stability of isorenieratene was compared with dietary carotenoid by mimicking the gastric conditions thus exhibiting good stability with the retention rate after ingestion and hence proved to have application in food (Chen et al. 2018). Apart from this Mukherjee et al. (2017) isolated Thermus strain from the water sample that inhibited protein oxidation activity and the pigments could be recommended for the food preservation application. Similarly, Kocuria sp. strain QWT-12 a halotolerant bacterium was isolated from tannery industry wastewater which produced a neurosporene pigment belonging to the carotenoid family with extraordinary antioxidant activity and UV-B radiation protector (Rezaeeyan et al. 2017). Blakeslea and Phycomyces are the best producers of antioxidant-rich lycopene which have various applications in dairy, snack food such as chips, soups, sweets to increase the shelf life and also used in the suppression of MSF-7 tumor cells proliferation. It was also used in the surimi and in fish processing industry

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especially for the salmon fish to intensify the pink color (Numan et al. 2018). The carotenoid from Pedobacter, Fontibacter flavus YUAB-SR-25 was reported to contain excellent oxygen scavenging properties with its defense mechanism hence can be used in the fatty-rich products to avoid peroxidation. Followed by violacein, pyomelanin, and carotenoid from Chromobacterium violaceum, Burkholderiaceno cepacia, Planococcus including flexirubin from Chryseobacterium artocarpi are the other antioxidant-rich pigments which have appreciable use in food industry (Venil et al. 2020a, b). In particular, violacein one of the protective pigments preventing age-related diseases and others such as ulcer, cataract, or degeneration of macular are prevented using microbial pigments (Nigam and Luke 2016). Bacillus VITPS12 and Planococcus maritimus VITP2 isolated from the mangrove forest were the richest source of multiple carotenoids and have therapeutic properties due to the presence of other functional groups (Prathiba and Jayaraman 2018).

9.4.3

Application in Cosmetic and Pharmaceutical Industry

Products produced with natural colors are preferred in spite of the health benefits; they also have essential clinical efficacy in the pharmaceutical industry and potential tools in the cosmetic industry. Numerous secondary metabolites from microorganisms have anticancer, antibiotic, antimalarial, and other beneficial properties. The major colorants from nature are prodigiosin, astaxanthin, and melanin in the cosmetic industry as they are the best source for UV protection. In general, biopigments are used in cosmetics either as sunscreen or as anti-aging agents. Pigments such as prodigiosin and violacein from S. marcescens and C. violaceum are used commercially in the cosmetic industry for the sunscreen application as they are naturally antibacterial with antioxidant capacity comparable to ascorbic acid (30% prodigiosin and 20% violacein) (Suryawanshi et al. 2015). For any sunscreen lotion, one of the key parameter is the sun protection factor (SPF). Choksi et al. (2020) isolated multiple organism from soil and water sample among which pigment from two strain (BUY1 and JFY) resulted with the highest SPF value (8.34 and 7.72). Similarly, Silva et al. (2019) Arthrobacter agilis, Arthrobacter psychrochitiniphilus and Zobellia laminarie produced UV-protective pigments that have the ability to withstand the UV-B and C radiation thus protecting the skin. These pigments are also called as photoprotective agents, most of the transbacterioruberin are used in the sunscreen. Apart from this Synechocystis pevalekii, a cyanobacterial pigment can also be used in the sun-protective formula (Tendulkar et al. 2018). Next most important pigment melanin, one of the major pigments for protecting the skin from disorders, is caused due to ultraviolet rays (Kageyama and WaditeeSirisattha 2019). Various sources of melanin include Aspergillus fumigates, Vibrio cholerae, Cryptococcus neoformans, Colletotrichum lagenarium, Alteromonas nigrifaciens, and most of the Streptomyces species (Narsing Rao et al. 2017). One of the significant producers of melanin was Streptomyces glaucescens NEAE-H (yield of 31.650 μg/0.1 mL) which was isolated from the soil sample exhibited a

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significant anticancer activity in skin through the in vitro MTT assay in skin cancer cell line while at increased concentration it showed low cytotoxicity. For this reason, it was used to minimize the tissue destruction in the cosmetics field (El-Naggar and El-Ewasy 2017). Pigment from Monascus has been widely used for the multiple fields starting antimicrobial, anti-mutagenic and even one of the wide known applications is its anti-obesity properties. With respect to the cosmetic industry, they were used for hair coloring due to its non-toxic nature but when this pigment is exposed to direct UV rays pigment discoloration is possible (Agboyibor et al. 2018).

9.4.4

Application in the Textile Industry

The concepts of health, organic, natural, and eco-friendly have turned people’s perception towards natural colors. Apart from increased marketability, the natural colors have antimicrobial property in spite of the wide range of ecological and medicinal properties. But the required organic substrate for growth of color producing microbes comes with rigorous efforts and is influenced by various process parameters (Tuli et al. 2015). A set of researchers conducted a study to isolate pigment producing fungi Talaromyces verruculosus strain from spoiled mangoes, and those red pigments were used in dyeing cotton clothes (Chadni et al. 2017). While in Kerala different soil samples were collected among which researchers isolated 37 Actinomycetes isolate were isolated as test organism for pigment production. The dyeing quality of the pigment were experimented in different cloth material while the uptake of color was good in synthetic fiber (Nair et al. 2017). Similarly, a yellow color pigment with 80% dyeing capacity was produced from Aspergillus sp. isolated from the soil sample (Pandiyarajan et al. 2018). Followed by this, Lagashetti et al. (2019) reported different colors such as yellow, green, and red from S. ganodermophthorum, C. aeruginosa, and S. cuboideum with good stability and colorfastness for dying wool, cotton, and spun with no toxic effect. The stringent environmental regulations increase the demand for bio-resources such as fungal pigments over synthetic pigments while commercialization of these pigments becomes tedious process due to the market regulatory affairs. Apart from this, Hernández et al. (2019) also reported the production of yellow and red dyes production from fungi isolated from the wood samples. Penicillium murcianum and Talaromyces australis pigments are used in the dyeing of woolen clothes at industrial level. With the greater potential of microbial pigment’s staining property, they are exploited through various researches to replace the non-renewable synthetic colorants. Extraordinary ranges of pigments obtained from different classes of fungi are biodegradable and sustainable and are comparatively advantageous to synthetic dyes. Yan et al. (2019) studied the yellow pigment from Metarhizium anisopliae for dyeing of silk and wool at optimum temperature between temperatures of 80 and 90
C under optimum dyeing time of 1 h. Followed by this, red pigment extracted from Rhodonellum psychrophilum was isolated from Tso Lake in India which is used in dyeing different fabrics. These fabrics were tested for

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the antibacterial and antifungal activity, apart from these studies have also reported for the antimicrobial activity of red and yellow color pigment from colored isolated from Serratia marcescens and Micrococcus luteus (Bisht et al. 2020). Among important industrial fermentation organisms Aspergillus and Trichoderma sp., were studied by Venugopal and Khambhaty (2020) for the pigment production using agro-industrial waste as a replacement for synthetic dyes. Apart from this, pigments from Penicillium minioluteum, Monascus purpureus, and Penicillium chrysogenum can be used for dyeing of leathers. Different pigment sources are used to dye different fabrics; pink color anthraquinones were used to dye cotton yarn extracted from Sclerotinia sp. also the pigments from Emericella nidulans, Monascus purpureus, Fusarium verticillioides, and Penicillium purpurogenum, Alternaria alternata, Isaria farinosa were used. Likewise pigment from Trichoderma sp. for silk fabric T. australis and P. murcianum red and yellow pigments was used to dye wool while (Venil et al. 2020a, b). For dyeing of wool, pigments from Chlorociboria species, Scytalidium cuboideum, Scytalidium ganodermophthorum, and Fusarium oxysporum were isolated, and they are also used for dyeing of cotton, polyester, nylon, etc. owing to their strong non-degradation property. The pigment quality was tested using different tests such as dipping, submersion, and saturation (Palomino Agurto et al. 2017). Dyeing properties are important factors for any industrial application of pigments hence sorption kinetics behavior, dyeing rate constant (k), and half-time of dyeing (t1/2) were measured. Apart from this concentration of the pigment, pH and temperature are imporatant in the optimization of dyeing of fabrics (Morales-Oyervides et al. 2017). For example, an orange color pigment isolated from soil Salinococcus roseus under the optimum condition of 40
C and pH 7, for 96 h had maximum pigment production with better stability (Usman et al. 2018). Multi-facilitated potency of prodigiosin pigment produced by Serratia could be explored not only for antibacterial and antifungal but also as an effective biocontrol agent in agriculture. Inoculation of bacterial isolate in peanut broth at 300
C for 72 h could yield maximum pigment production and the purity of the pigment extracted was confirmed by the spectrophotometric method and by TLC. When compared with the standard Nistatin, MIC of the pigment was observed at 80 μg for Alternaria and for Fusarium MIC was observed at 160 μg. Insecticidal activity of 100% mortality was found against cockroaches and tropical ants while 85–71% was observed with termites and pyramid ants (Sagar et al. 2019).

9.5

Conclusion

Exploration of microbial pigments to obtain the natural colorants and its possible application is the need of the day. Novel insights regarding the source of pigmented organisms and the biotechnological tools used to optimize the production to pave way for multifaceted roles creates impact in the current era. From the dietary inclusion, textile dyeing, and pharmaceutical application reveals its scalability in the industrial scale.

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Acknowledgments One of the authors, Selvaraju Vishnupriya acknowledges TANUVAS for providing PhD fellowship and for the facilities provided by College of Food and Dairy Technology, TANUVAS. Conflict of Interest The authors declare no conflict of interest. Consent for Publication Not applicable.

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Pandiyarajan S, Premasudha P, Kadirvelu K (2018) Bio-production of novel water-soluble yellow pigment from Aspergillus sp. and exploring its sustainable textile applications. 3 Biotech 8 (9):398 Panesar R, Kaur S, Panesar PS (2015) Production of microbial pigments utilizing agro-industrial waste: a review. Curr Opin Food Sci 1:70–76 Park SA, Ahn SY, Choi KY (2020) Functional microbial pigments isolated from Chryseobacterium and Deinococcus species for bio-paint application. Biotechnol Bioprocess Eng 25(3):394–402 Patthawaro S, Lomthaisong K, Saejung C (2019) Bioconversion of agro-industrial waste to valueadded product lycopene by photosynthetic bacterium Rhodopseudomonas faecalis and its carotenoid composition. Waste Biomass Valorization 11:2375. https://doi.org/10.1007/ s12649-018-00571-z Pereira da Costa D, Campos Miranda-Filho K (2019) The use of carotenoid pigments as food additives for aquatic organisms and their functional roles. Rev Aquac. https://doi.org/10.1111/ raq.12398 Prathiba S, Jayaraman G (2018) Evaluation of the anti-oxidant property and cytotoxic potential of the metabolites extracted from the bacterial isolates from mangrove Forest and saltern regions of South India. Prep Biochem Biotechnol 48:750–758. https://doi.org/10.1080/10826068.2018. 1508038 Ram S, Mitra M, Shah F, Tirkey SR, Mishra S (2020a) Bacteria as an alternate biofactory for carotenoid production: a review of its applications, opportunities and challenges. J Funct Foods 67:103867 Ram S, Tirkey SR, Kumar MA, Mishra S (2020b) Ameliorating process parameters for zeaxanthin yield in Arthrobacter gandavensis MTCC 25325. AMB Express 10:1–13 Rezaeeyan Z, Safarpour A, Amoozegar MA, Babavalian H, Tebyanian H, Shakeri F (2017) High carotenoid production by a halotolerant bacterium, Kocuria sp. strain QWT-12 and anticancer activity of its carotenoid. EXCLI J 16:840 Sadh PK, Duhan S, Duhan JS (2018) Agro-industrial wastes and their utilization using solid state fermentation: a review. Bioresour Bioprocess 5:1–15 Saejung C, Puensungnern L (2020) Evaluation of molasses-based medium as a low cost medium for carotenoids and fatty acid production by photosynthetic bacteria. Waste Biomass Valorization 11(1):143–152 Sagar BSV, Deepak BS, Tejaswini GS, Aparna Y, Sarada J (2019) Evaluation of prodigiosin pigment for antimicrobial and insecticidal activities on selected bacterial pathogens & household pests Salvo A, Giuffrida D, Rotondo A, Pasquale PD, La Torre GL, Dugo G (2017) Determination and quantification of carotenoids in sea sponges Raspacionaaculeata and Dictyonella marsilii present in the Ganzirri Lake (Messina), Italy. Nat Prod Res 31(20):2397–2404 Sehrawat, R., Panesar, P. S., Panesar, R., & Kumar, A. (2017). Biopigment produced by Monascus purpureus MTCC 369 in submerged and solid-state fermentation: a comparative study. Pigm Resin Technol, 46:425-432 Sharma R, Ghoshal G (2020) Optimization of carotenoids production by Rhodotorula mucilaginosa (MTCC-1403) using agro-industrial waste in bioreactor: a statistical approach. Biotechnol Rep 25:e00407 Silva TR, Tavares RS, Canela-Garayoa R, Eras J, Rodrigues MV, Neri-Numa IA et al (2019) Chemical characterization and biotechnological applicability of pigments isolated from Antarctic bacteria. Mar Biotechnol 21(3):416–429 Suryawanshi RK, Patil CD, Borase HP, Narkhede CP, Stevenson A, Hallsworth JE, Patil SV (2015) Towards an understanding of bacterial metabolites prodigiosin and violacein and their potential for use in commercial sunscreens. Int J Cosmet Sci 37(1):98–107 Taufiqurrahmi N, Religia P, Mulyani G, Suryana D, Ichsan Tanjung FA et al (2017) Phycocyanin extraction in Spirulina produced using agricultural waste. IOP Conf Ser Mater Sci Eng 206:012097

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Part II Microbial Polymers in Agriculture

Extracellular Polymeric Substances from Agriculturally Important Microorganisms

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Valeria Valenzuela Ruiz, Roel Alejandro Chávez Luzania, Fannie Isela Parra Cota, Gustavo Santoyo, and Sergio de los Santos Villalobos

Abstract

Within microorganisms, there is a group capable of producing extracellular polymeric substances (EPS), which are proteins, polysaccharides, lipids, nucleic acids, and humic substances, produced and excreted outside the cell. Microorganisms have EPS to facilitate their vital metabolic processes, these substances are necessary to carry out chemical reactions within their natural habitat to trap nutrients or protect the plant against abiotic factors such as salinity and drought. Thus, their use in agriculture to promote plant growth and health has been widely studied. EPS have also been a subject of interest in agriculture due to their abilities to improve soil quality, increase soil aggregates, and contribute to improve soil fertility. In addition, use of EPS in soil surrounding plant roots promote benefits in availability of water in contact with plant and in nutrient absorption, these benefits affect both host and surrounding microorganisms, thus creating a more suitable ecosystem for plant developement. These benefits were observed through EPS producer inoculation in wheat, chickpea, and sunflowers, among other crops of agronomic importance.

V. Valenzuela Ruiz · R. A. Chávez Luzania · S. de los Santos Villalobos (*) Instituto Tecnológico de Sonora, Ciudad Obregón, Sonora, Mexico e-mail: [email protected] F. I. Parra Cota Campo Experimental Norman E. Borlaug, Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Ciudad Obregón, Sonora, Mexico G. Santoyo Instituto de Investigaciones Químico Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, Mexico # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_10

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Keywords

Plan growth-promoting microorganisms · Agriculture · Soil health · Beneficial bacteria

10.1

Introduction

Food is one of the most important basic needs for human beings; in turn it has an important implication on society at an economic and personal development level (Etesami and Maheshwari 2018). As technology and knowledge about the food we consume advances, increasing strict parameters have been established for the quality of these products, governmental and competent international organizations have turned to see safe and sustainable food production as a priority (Kenneth et al. 2019). In addition, the population increase around the world also results in an increase in the demand for not only more food production but also at higher quality. It is estimated that there will be around 10 billion people on the planet by the year 2050 (FAO 2017). To meet this food demand, production must increase by roughly 70% by 2050 and double or triple by 2100 (Crist et al. 2017). Currently, in industrialized agricultural techiques predominate practices that consume fossil fuels and require excessive applications of fertilizers that not only harm soil and crops, but also alter microbial communities that play an important role in plant development; in addition to contaminating the surrounding environment, resulting in i.e. saline and compacted soils and water contamination (More et al. 2014). Although some of these practices have immediate advantages in agricultural production, their effects are perpetuated in the environment, causing a problem that ends up affecting the quality of life for humans and other species, as well as long term production (Kenneth et al. 2019). Due to the population increase, the demand for food and the need to generate quality products, research should focus on the development of new methods with sustainable approaches to meet the current food demand (Etesami and Maheshwari 2018). Plants interact with ~1  109 microbial cells g 1 dry soil and 1  105 microbial species g 1 dry soil (Valenzuela-Aragon et al. 2019). Microorganisms and microbial communities that are found in the phyllosphere [total plant proportion as a habitat for microorganisms, compromising the aerial parts of plants and is dominated by the leaves, where the global bacterial population present could comprise up to 1026 cells and fungal population is yet to be estimated (Vorholt 2012)] and the rhizosphere [the soil portion that is influenced by root exudates, where the microbial community residing in this niche is driven by the direct effect of the presence of root exudates, leading to increased microorganisms biomass; and high nutrient availability, which have the ability to change environmental conditions on rhizosphere, creating a microhabitat, whose concentration can be up to 1012 cells/g of soil (Brink 2016; Andreote et al. 2014)] in agro-ecosystems, are being targeted by their potential for sustainable agriculture in search of new alternatives to obtain better yields in agriculture and contribute to food security (Valenzuela-Aragon et al. 2019; de los Santos-Villalobos et al. 2018).

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The interaction of microorganisms with plants forms a complex network of metabolite exchange, through which biological mechanisms are activated or repressed (Kenneth et al. 2019). Some of these mechanisms, described later, provide microorganisms plant growth promotion and/or biological control, depending on the species, regulated by environmental factors, nutrient availability, and the plant– microorganism interactions (Yadav et al. 2020). Plant growth-promoting microorganisms (PGPM), mainly bacteria (PGPB), promote plant development and overall health, allowing it to grow optimally by reducing stress conditions around it (Rojas-Padilla et al. 2020). On the other hand, microorganisms that have the ability to act as antagonists against pests that affect crops have been classified as biocontrol agents (BCA) (Orozco-Mosqueda et al. 2018). Within the complex network of metabolites generated by microorganisms in the vicinity of the plant, there is a group whose main differentiator is its polymeric nature, the extracellular polymeric substances (EPS). These substances have diverse functions which are useful for modern agriculture, some PGPB and BCA are EPS producers (Siddiqui and Khan 2017; Valenzuela Ruiz et al. 2020). Thus, in this chapter an overview of the current agronomic researches on EPS are discussed.

10.2

Plant Growth-Promoting Bacteria

This is assigned to those bacteria capable of causing a beneficial effect on plants, they are mainly found in soil, plant root surface up to a distance of 5 mm (and is related to the plant genotype, edaphic-climatic conditions, among others), and within their own tissues (Lakshmanan et al. 2017; Rodriguez et al. 2019). These microorganisms have stimulating mechanisms (1) indirect mechanisms, plant protecting against adverse factors by the production of antibiotics and lytic enzymes, as well as the synthesis of hydrogen cyanide and volatile compounds (Orozco-Mosqueda et al. 2018); or (2) direct mechanisms, which involve either improving their development through the modulation of hormones or facilitating the acquisition of essential nutrients for the plant by phosphorus, potassium, and zinc solubilization, the biological fixation of nitrogen and the siderophores production, ammonia and secondary metabolites such as EPS with growth-promoting properties (Table 10.1) (de los Santos-Villalobos et al. 2018; Yadav et al. 2020; ValenzuelaAragon et al. 2019). EPS-producing microorganisms are capable of establishing interactions with plants in a way that promotes their development, stimulating microorganisms present in the surrounding soil community and exudate production, where organic carbon released by roots surfaces favors microbial community proliferation in the rhizosphere, which in turn, promotes soil aggregation, producing mucilaginous EPS, and increasing the amount of soil attached to the roots (Costa et al. 2018). Soil aggregation in the roots by the action of EPS form a favorable plant growth environment with nutrient and water retention properties (Rashid et al. 2016). Likewise, certain microorganisms have biocontrol actions that protect the plant against different microorganisms (Table 10.2) (de los Santos-Villalobos et al. 2018; Yadav et al. 2020;Valenzuela-Aragon et al. 2019).

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Table 10.1 Bacteria that promote plant growth in agro-ecosystems PGPB Rhizobium leguminosarum Pseudomonas putida

Crop Direct growth promotion of canola and lettuce Early developments of canola seedlings

Azospirillum brasilense and A. irakense P. fluorescens

Growth promotion of wheat and maize plants Growth of pearl millet

P. putida strain

Tomato plant growth improvement

Azospirillum and Azotobacter

Canola plant growth and productivity improvement

Pseudomonas aeruginosa and Bacillus firmus

HCN production, NH3 production, Siderophore production, antagonist, and phosphate solubilizer Chick pea growth stimulation

Azotobacter, Azospirillum, and Pseudomonas Pseudomonas sp. and R. leguminosmarum P. putida

P. putida and fluorescens, A. brasilense, and lipoferum P. fluorescens Bacillus sp. and Burkholderia sp. Bacillus polymyxa, P. alcaligenes, and Mycobacterium phlei Rhodococcus sp., Pseudomonas sp., and Arthrobacter nicotinovorans Azospirillum brasilense and Bradyrhizobium japonicum

Improve yield and phosphorus uptake in wheat Seed germination, growth parameters of maize seedling in greenhouse and also grain yield of field grown maize Improves seed germination, seedling growth, and yield of maize Increase growth, leaf nutrient contents, and yield of banana Phosphate solubilizer N, P, and K uptake improvement by maize crop

Reference Noel et al. (1996) Glick et al. (1997) Dobbelaere et al. (2002) Niranjan et al. (2003) Gravel et al. (2007) Yasari and Patwardhan (2007) Sandilya et al. (2016, 2017) Rokhzadi et al. (2008) Afzal and Asghari (2008) Gholami et al. (2009) Nezarat and Gholami et al. (2009) Kavino et al. (2010) Oliveira et al. (2009) Egamberdiyeva (2007)

Phosphate solubilizer

Sofia et al. (2014)

Phytostimulation

Cassan et al. (2009)

The previous tables show success cases where the inoculation of PGPB recognized as EPS producers showed better plant development, i.e., P. fluorescens Pf1 and P. fluorescens CHA0 (Kavino et al. 2010) have showed growth promotion in banana cultivation and Bacillus subtilis strain TE3T (Villa-Rodriguez et al. 2019) has showed biocontrol against Bipolaris sorokiniana in wheat.

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Table 10.2 Biological control agents used in crop production BCA Bacillus pumilus, Kluyvera cryocrescens, B. amyloliquefaciens, and B. subtilis B. amyloliquefaciens, B. subtilis, and B. pumilus B. pumilus

Crop Cucumber mosaic virus (CMV) of tomato Tomato mottle virus

Burkholderia

Bacterial wilt disease in Cucumber Downy mildew in pearl millet Foliar diseases of tomato Maize rot

Bacillus subtilis

Spot blotch in cereals

B. subtilis, B. pumilus R7, and B. pumilus B. cereus

Reference Zehnder et al. (2000) Murphy et al. (2000) Zehnder et al. (2001) Niranjan et al. (2003) Silva et al. (2004) Hernandez Rodriguez et al. (2008) Villa-Rodriguez et al. (2019)

The main hypothesis to explain the efficiency of colonization by PGPB or biological control agents is the chemotaxis effect (Yadav et al. 2020). Plant colonization by bacteria and fungi depends on their ability to (1) move toward the root, (2) use nitrogen and carbon sources contained in soil, (3) or those given by exudates of root to shape the root microbiome, (4) resist plant’s response reaction, including reactive oxygen species (ROS), and (5) have the ability to form biofilms on the root surface (Kumar and Singh 2020). The study of the behavior of PGPB in agroecosystems has shown a wide range of action mechanisms to achieve their activities, where EPS are an important group of biomolecules produced for these purposes. In addition, these substances promote the production of beneficial biomolecules for plant development or, failing that, the mitigation of stress-causing biomolecules (Parra-Cota et al. 2018).

10.3

Extracellular Polymeric Substances

PGPB are able to produce diverse groups of EPS; these are composed of proteins, nucleic acids, polysaccharides, humic substances, and lipids (Fig. 10.1). Thus, EPS are produced and secreted into the extracellular environment, either because they are essential for life or due to the persistence of microorganisms, because they give an ideal condition to carry out chemical reactions since they trap nutrients or protect against abiotic factors such as drought and salinity (More et al. 2014). The biomolecules included within EPS have a diverse group of compounds that vary their effect on plants according to their nature and chemical composition (Flemming et al. 2016). EPS are molecules that are produced in exchange for high energy consumption in cells; they are mainly synthesized by environmental signals and provide an advantage comparable to the energy expenditure used (Flemming et al. 2016). The EPS, as

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Fig. 10.1 Main EPS produced by bacteria and their composition. Polysaccharides, proteins, nucleic acids, lipids, and humic acids have the ability to improve bioabsorption and soil aggregates although to a greater extent the first two groups

a resource, are important in different ecosystems since they allow the permanence of certain beneficial organisms in the niche. Agro-ecosystems are an important deposit for EPS producers where they provide advantages to crops (Costa et al. 2018). EPS composition grants it important properties for agricultural uses, such as nutrient entrapment; protection against drought and salinity stress; as well as heat and cold stress; antimicrobial protection; better soil texture; and generating symbiosis with plants where these interactions favor soil and plant fertility, resilience and overall plant health (More et al. 2014). These substances differ from each other by their physical and chemical properties of each type of polymer, i.e., (1) polysaccharides have adhesion, cell aggregation, increased water retention, adsorption properties of organic and inorganic compounds, they form a protective barrier, and are a source of nutrients, (2) proteins possess adhesion, cell aggregation, water retention properties, binding of enzymes, acceptor and electron donor, absorption of organic and inorganic matter, and provides a protective barrier for cells, (3) nucleic acids possess properties of adhesion, aggregation of cells, source of nutrients, exchange of genetic information, export of cellular components, (4) lipids differ by being exporters of cellular components, and (5) humic substances provide adhesion, being electron acceptors and donors (More et al. 2014). The EPS matrix serves as a reservoir of extracellular enzymes which capture substances from the aqueous phase and favors that these compounds are used as a source of energy, retaining carbon, nitrogen and phosphorus containing compounds (Bach et al. 2010; Flemming and Wingender 2010). During the course of biofilm maturation, soluble EPS forms a filamentous matrix that maximizes nutrient distribution and speeds up the cell aggregation (Berk et al. 2013). EPS have the ability to

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generate cohesion between bacteria, causing adhesion of biofilms to surfaces, modify spatial organization, permitting the exchange of metabolic information between microorganisms, and allowing adhesion between them; therefore, establishing a stable consortium of mixed population communities, and hence, diverse synergistic relationships (Laspidou and Rittman 2002; Flemming and Wingender 2001). An example is the nitrification process, which takes place in biofilms and allows the spatial closeness of ammonia oxidizers to nitrite oxidizers (Wan et al. 2013). Also, different protein (adhesins) and extracellular DNA form EPS are related in the process of adhesion of microorganisms to plant surfaces, these being beneficial traits for plant colonization of plant growth-promoting microorganisms (Okshevsky and Meyer 2013; Wan et al. 2013). On the other hand, amino acids present in EPS function as both carbon and nitrogen sources for the root-inhabiting bacteria; for example, agriculturally important bacteria such as Pseudomonas fluorescens and Bacillus subtilis showed motility toward proteinogenic amino acids which some EPS present (Velmourougane et al. 2017). Nutrient retention and root-inhabiting facility is important for plant growth and health where the retention or availability of nutrients is crucial for plant development (Yadav et al. 2020). Thus, EPS are considered a viable alternative to replace conventional agrochemicals, standing out for their biodegradability, high efficiency, non-toxicity, and no generation of secondary pollutants (More et al. 2014). Currently, the development of production methods for substances capable of promoting greater plant yield and microorganisms that produce them are of research interest. In recent years, the understanding of the mechanisms involved in plant–microorganisms interaction is determinant to modulate the microbial population in soil for increasing food production.

10.4

Agricultural Important Roles of EPS

10.4.1 Symbiosis A mechanism widely studied in EPS is its function in symbiosis relation among atmospheric nitrogen-fixing rhizobacteria and plants, where polysaccharides, key in formation of nodules (Costa et al. 2018). Van Workum et al. (1998) stated that EPS produced by R. leguminosarum accelerate the root hair curling rate and infection to extent the rhizobial root penetration in Vicia sativa. In a recent study, the relation between EPS of Lotus japonicus and Mesorhizobium loti regulated by a receptor expressed by the plant was investigated, which permits infection of bacteria that produce EPS (Kawaharada et al. 2015). The expression of this receptor shows how the plant can identify EPS structures produced by rhizobial microorganisms. The importance of beneficial relations established between the soil, plant, and microbiota is key for optimal crop production; however, one of the main limitations is crop yield loss due to plant pathogens.

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10.4.2 EPS as Pathogenicity/Virulence Factors Every year around the world, biotic stress in crops caused by diseases, weeds, and insects causes the loss of 31–42% of food production, which represents 500 billion dollars; in addition to an extra loss of 6–20% (120 billion dollars) due to fungi, bacteria, and insects after harvest. These losses tend to be aggravated when dealing with developing countries, where the percentages in comparison with developed countries differ up to 22% (Shameer et al. 2019). EPS are able to protect plants against pathogens, presenting biologic control activity; however, they may also function as a mechanical wall among phytopathogens and plant defenses (Velmourougane et al. 2017). For instance, Pseudomonas syringae pv. phaseolicola and S. meliloti polymers are able to protect bacteria against reactive oxygen species that are produced by the plant host during infection, resulting in an oxidative stress decrease (Lehman and Long 2013). Similarly, to Agrobacterium, Bacillus clavibacter, Erwinia, Pantoea, Pseudomonas, Xanthomonas, and Ralstonia genere (Caro-Astorga et al. 2014;Merritt et al. 2007; Tomlinson et al. 2010; Vlamakis et al. 2013; Ramey et al. 2004).

10.4.3 Drought Stress In many agriculturally important microorganisms, EPS are really relevant in abiotic and biotic environmental stress combat, e.g., drought, temperature, salt tolerance, and phytopathogens (Bogino et al. 2013). The matrix made of EPS is capable of supporting up to 20 times its weight in water (Flemming and Wingender 2001) and contributes to soil moisture (Roberson et al. 1993). Biofilms are able to hold up water in microenvironments either directly owing to its hygroscopic properties, or for its ability to modify the conformation of the biofilm, an ability that helps to avoid the loss of water by evaporation (Costa et al. 2018). Evaporation losses are not the only way water is retained (Mager 2010) in a study conducted by Cho et al. (2018). They demonstrated that the EPS of P. chlororaphis had a positive effect against drought; they caused a limited wilting and improved the water retention capacity, thus increasing the photosynthetic efficiency in Arabidopsis plants. Similarly, Timmusk and Zucca (2019) stated that biofilm formation is required in induction of drought tolerance. As a result, the EPS matrix makes microbial cells more tolerant to drought conditions and thereby prolonging their survival rate and residence time in soils a beneficial trait for plant growth-promoting microorganisms (Velmourougane et al. 2017).

10.4.4 Heat Stress Among the most important stress conditions is stress due to high temperatures, which constitutes one of the conditions with the greatest impact on crops, since heat modifies water relations, the rate of transpiration, the balance of photosynthesis,

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efficiency of the use of water, protein synthesis and enzymatic activity; this causes burns in branches and leaves, senescence of leaves, less growth, and discoloration of leaves and fruits, which consequently causes a decrease in agricultural yield (Krieger-Liszkay et al. 2018). In relation to the above, EPS have shown positive effects in the improvement of plant growth in a situation of heat stress, these are a protection factor for microorganisms exposed to places with high temperatures, which allows them to proliferate and develop in such a way that it can continue with its metabolism and carry out the promotion of plant growth, thus the EPS act indirectly on the plants for their benefit (Sengupta and Dey 2019). In addition to the fact that the EPS matrix has important functions as a diffusion barrier to antimicrobial agents, it also provides protection against heat stress; this matrix favors the retention of water in the vicinity of the plant, which allows it to have this resource available for its development, it is in this way that growth promotion occurs in response to abiotic stress (Harimawan and Ting 2016).

10.4.5 Salt Stress A growing problem in current agriculture are saline soils, where world estimates show that stress from saline conditions is increasing, particularly in irrigated areas, where the augmentation was from 45 million hectares to 62 million hectares between 1990 through 2013 (Qadir et al. 2014). Salinity causes harmful effects on plant growth; this affects the quality of water for agronomic uses, is a factor of soil erosion, and affects sedimentation. Furthermore, EPS have the ability to improve tolerance to saline stress, both for plants and for EPS-producing microorganisms associated with them,(Costa et al. 2018). Bacterial EPS can bind cations including Na+ thereby alleviate salinity stress by decreasing the levels of Na+ in the surrounding environment available to the plant. There are also reports of EPS-producing plant growth-promoting rhizobacteria to significantly enhance the volume of soil macropores and the rhizosphere soil aggregation, resulting in increased water and fertilizer availability to inoculated plants (Upadhyay et al. 2011). Salt-tolerant ACC-deaminase containing PGPB can alleviate soil salinity stress during plant growth by reducing ethylene synthesis while bacterial EPS will ensure its better survival in field and can also help to mitigate salinity stress by reducing the concentration of Na+ in soil (Kumari and Khanna 2015). An example of this mechanism is reported by Kumari and Khanna (2015), in chickpea (Cicer arietinum L.) growth promotion by the inoculation of EPS-producing rhizobacteria from genera Enterobacter Burkholderia, Achromobacter, Bacillus, and Pseudomonas. Similarly, Arora et al. (2010) stated that EPS produced on germination of maize, wheat, and rice by a consortium of cyanobacteria were studied at different concentration of salt. EPS production was found to be stimulated by salts, which in turn had a significant Na+ removal capability from aqueous solution, and the germination of seeds, vigor index, and efficiency of mobilization in all the three crops improved when cyanobacterial EPS were applied.

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10.4.6 EPS and Soil Structure Soil structure plays a fundamental role in ecosystem functions which is crucial for plant development, where nutrient availability, water retention, and the presence of microbial community is dependent of good soil structure, therefore, regulating soil fertility and plant productivity (Vezzani et al. 2018). EPS related to microorganisms present in soils represent 0.1–1.5% of soil organic matter (Chenu 1995). According to Chenu (1993), typical EPS values found in soils (assuming 109 cells g 1 of soil with total weight of 1.1 mg) ranges between 0.1 and 5.0 mg EPS (or 10–500% of cells’ biomass) (Or et al. 2007). Also, EPS produced in situ is expected to improve soil properties, for example, increasing heterogeneity (Davis et al. 2009) and improving soil aggregate stability (Tang et al. 2011). Aggregates in soils are of great importance to improve various characteristics such as erodibility, porosity, agronomic performance, and root elongation. (Bronick and Lal 2005). Evidence of EPS-producing microorganisms involved in soil quality (Table 10.3) are, for example, Paenibacillus polymyxa which was implicated in the aggregation of rootadhering soil on wheat (Bezzate et al. 2000); Pantoea agglomerans regulates rhizosphere’s soil water content by improved soil macroaggregation (Kaci et al. 2005); Rhizobium modifies the soil structure around a sunflower root system after inoculation, counteracting the negative effect of a water deficit on plant growth (Alami et al. 2000); P. putida is capable of improving soil stability by 150% in salinity, high temperatures, and drought conditions (Vardharajula and Ali 2015), Bacillus amyloliquefaciens, B. licheniformis, and B. subtilis improve aggregate stability under drought stress (Vardharajula and Ali 2014). The production of EPS in cells is given by the internal and external factors that surround them; thus, the factors delimiting production are: genotype, physical, chemical, and biological Table 10.3 Main producers of EPS and their main benefits to soil aggregation Strain P. putida

Paenibacillus

Bacillus amyloliquefaciens, B. licheniformis, and B. subtilis Microbacterium arborescens N. muscorum

Stachybotrys atra

Benefit Improvement of the stability of soil aggregates by more than 50% in conditions of stress due to salinity, drought, and temperature Promote the growth of bacterial heterotrophic community and increase its population, in addition to increasing available N and P Aggregate stability improve in stress absence and under drought stress It cause a strong binding in sandy soil better aggregation in a sandy soil than in agricultural Improves soil aggravation by 18% and increases total soil carbon by more than 60% and nitrogen by more than double Increase the aggregation of fumigated soil

Reference Vardharajula and Ali (2015) Wu et al. (2014) Vardharajula and Ali (2014) Godinho and Bhosle (2009) Rogers and Burns (1994) McCalla et al. (1958)

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factors, in addition to the carbon / nitrogen ratio, pH, and temperature (More et al. 2014).

10.5

Inoculation of EPS Producers in Crops

Few of the main problems we are currently facing due to traditional agricultural practices are compacted and saline soils, as well as inefficient crop yields in relation to the amount of agro-chemicals applied, and soil degradation (Lares-Orozco et al. 2016; Herrejón 2014; Miransari 2011; Dotterweich 2013). This last one has impacted 80% of agricultural soils worldwide (Nkonya et al. 2015). EPS inoculated in plants surrounding soils improve water retention, dampen changes in the water potential, promote production of root exudates, and improve nutrient uptake and plant growth. The improvement in soil aggregation and structure is able to promote soil growth because it improves the nutrients absorption and maintains a good humidity degree in soil (Alami et al. 2000; Bezzate et al. 2000; Sandhya et al. 2009). In soil there are different components that promote its aggregation, and microorganisms are essential for soil aggregation. The extent to which microorganisms have in this regard depends on the diversity of species present, the available substrates, and soil management in that region (Costa et al. 2018). Bacillus, Pseudomonas, Rhizobium, and Pantoea stand out for being EPS producers and plant growth promoters; these microorganisms can be inoculated both in the soil and in the seedlings (Cipriano et al. 2016). In addition to pure cultures of microorganisms, the combination of these can be an interesting option to achieve a better effect on the soil and plants; however, there are not enough studies on the use of EPS-producing microorganisms consortia.

10.5.1 EPS Application in Agriculture The inoculation of PGPB is a very widespread and important practice in agriculture due to its multiple benefits; minimal changes in water potential increases nutrient absorption and improves plant growth. Thus, these microorganisms can provide multiple benefits to the plant, and their segregated EPS have the capacity to solve productivity problems and soil aggregation. For example, Tewari and Sharma (2020) carried out field experiments on Cajanus cajan using Rhizobium and its purified EPS, specifically exopolysaccharides. The experiment was conducted in northern India under natural stress: pH 8.9, 45  C, and 33 ppm NO3-N. The test was performed with and without the recommended dose of fertilizer in the form of diammonium phosphate, and in combination with treatments of the microorganism with and without EPS. All treatments significantly improved germination, pod number, seed yield, and protein content by 114, 138, 131, and 137%, respectively, compared to treatments without these two variables (control) (Tewari and Sharma 2020).

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In addition, Daud et al. (2019) reported the importance of Paenibacillus polymyxa in agriculture and its applicable properties in modern biotechnology focused in crops, for example, tomato (Cheng et al. 2017) maize (Sheela 2013) and rice (de Souza et al. 2015). This bacterium has been noted for its ability to fix atmospheric nitrogen, solubilize phosphates, the production of phytohormones and for the production of important EPS, such as (1) D-glucuronic acid for biofilm formation; (2) production of levanos and β-glucans as antioxidants and antitumor; (3) dehydrogenase and polysaccharide beads for the absorption of heavy metals; (4) polymyxin E1 and E2 as a bactericide; and (5) polymixin B as an antimicrobial (Daud et al. 2019). Similarly, Rhizobium is a wheat growth promoter; it has been shown that the EPS production of this strain improves plant growth, increases the soil attached to the root, and an increase in the amount of soluble aggregates is present in the water due to the decrease in soil water stress. It was found that the polymeric substance produced by this microorganism is composed of glucose, galactose, and mannuronic acid in a molar ratio of 2:1:1 respectively, this was observed when glucose, fructose, mannitol, and sucrose were used as carbon sources; later, it was determined by means of 1D and 2D NMR spectroscopy that consists of a repeating tetrasaccharide unit; this composition also provides polyelectrolytic properties (Kaci et al. 2005). Another case of success in the use of EPS to improve the productivity of crops is that of inoculation with Halomonas variabilis and Planococcus rifietoensis, both EPS producers, in chickpea plants. These strains were found to have the ability to form biofilms and accumulate exopolysaccharides by increasing saline stress, this allowed them to tolerate salinity, improved plant growth, and increased soil aggregation by more than 75% under high salt concentrations. These results showed that bacteria could be involved in the development of microbial communities under saline stress; in addition, evidence suggests that these strains are useful in the colonization of bacteria in plant roots and soil particles (Qurashi and Sabri 2012).

10.6

Conclusions and Perspectives

EPS are compounds with functions as diverse and complex as their structures. EPS play a relevant role in agricultural production; however, it is necessary to extend its study in other taxonomic groups that have not been profoundly studied and that are part of the plant microbiome. Likewise, it is required to know the interactions between the different EPS and their possible synergistic actions, either through the co-inoculation of mixtures of pure compounds or microbial strains that produce multiple EPS. Therefore, despite the actual and wide knowledge of EPS, it is also important to continue exploring on the identification of new polymers and potential benefits on plant crops, in order to innovate the bioinoculant formulation for increasing the food production, under current or future scenarios.

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Significance of Bacterial Polyhydroxyalkanoates in Rhizosphere

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Sundaresan Bhavaniramya, Selvaraju Vishnupriya, and Dharmar Baskaran

Abstract

Polyhydroxyalkanoates PHAs are groups of polyesters synthesized from different types of microorganisms utilized for production of biopolymers. Rhizospheres from soil produce PHA developing microbes thereby improving the ecological balance. Significance of producing bacterial PHA in rhizospheres has developed interest over the years. Global demand for replacing synthetic plastics through bio-based plastics and biodegradable polymers are widely in focus. Hence, the least expensive, universally available method of producing bacterial-based PHA for biopolymer manufacture can be optimized and established through soil rhizobium. The knowledge on bacterial PHA synthesis from soil rhizosphere has been advantageous and provides a wide range of applications including textile, packaging and polymer, food products packaging designs industries, etc. Recent approaches in developing bacterial PHA from different sources have been discussed in this chapter. Keywords

Biopolymer · Bacterial PHA · Rhizosphere · Soil

S. Bhavaniramya (*) · S. Vishnupriya College of Food and Dairy Technology, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India D. Baskaran Madras Veterinary College, Tamil Nadu Veterinary and Animal Sciences University, Chennai, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_11

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Introduction

PHAs are groups of bio-polyesters synthesized from various range microorganisms, viz., Gram-positive, Gram-negative bacteria. Commonly, PHAs are classified based on short chain length PHA containing C4-C5 monomers and medium chain length with C6-C24 monomers. The functions of PHA play a strategic role in the survival characteristics of microbes under varied environmental conditions. They are produced under limited conditions with excess carbon for the utilization in bioplastic production. Rhizospheres are defined as distinct soil compartments with varied organic acids, sugars representing the biology of soil. The rhizosphere microbiome enhances plant nutrition by nitrogen fixation and aiding uptake of plant nutrients. The interest for bacterial PHA from the rhizosphere focuses sustainable development of an eco-friendly environment. The biological origin of the polymers makes it biocompatible, biodegradable describing under green plastics. PHAs are accumulated by several bacteria in the form of intercellular granules that serve as a reservoir of energy. PHA was first discovers in 1925 by French Scientist Lemoigne in Bacillus megaterium in the form of (3-polyhydroxybutyrate) PHB (Chee et al. 2010). Generally, bacteria store PHA in the cytoplasm to require nutrients. Bacteria vary accordingly with PHA production, nutrient limiting bacteria accumulate PHA and those that do not accumulate during growth phase. For example, Pseudomonas putida, Pseudomonas oleovorans belong to the former group and recombinant E. coli to the latter (Muhammadi et al. 2015). Subsequent changes in soil organic matter actively monitored and that are associated with certain variations in the soil conditions. The bacterial PHA produced from rhizosphere soil bacteria involves biodegradable, economically effective polymers for the environment. Abundance and global dependence on synthetic plastics is a serious problem, further, waste management concerns on petroleum-derived plastics, petrochemicals cause threat to the environment. Generally, the production process is tedious, and recycling of synthetic plastics remains a challenging, time-consuming method. In such cases, biodegradable polymers, plastics from bacterial sources are the best solution to environmental hazards. The interest towards bacterially derived PHA has been settled for many years, and the cost of production along with slow growth of microorganisms possess a remarkable concern. Several industrial wastes are focused on candidates for PHA production, and PHA remains an obvious alternative to synthetic plastic wastes. PHAs are being produced industrially and cover broad spectrum end products from food packaging to medical applications. Regardless of the quality characteristics and advantages to the environment, PHAs are impaired for high cost of production (Amaro et al. 2019). The importance of high cost of production implies the use of costly fermenters for carbon source production. Thus, soil bacteria are ideal sources for carbon development and storehouse of nutrients. Rhizospheres from soil organic matter are significant producers of PHA solving environmental problems. The function of PHAs is storage reservoir of carbon utilized by microorganisms whenever required and that are generally present in the form of granules. Currently, PHAs are used as a blend form or in pure form covering a variety of applications: PHAs (short chain length) are often used in food

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packaging and disposable items, and PHAs (medium chain length) are applicable for high value-added applications, viz., materials for suture, surgical implants, etc. Industrialization and global usage of PHAs is limited due to high costs of production that improves the prices of petrol-based polymers, from polypropylene (PP) and polyethylene (PE). Further, industrialized the production of PHAs, have problems with the purity of substrates, and concerns of some carbon sources identified from human food supplies, and hence huge amounts of solvents required for the extraction in large-scale synthesis and makes the production process complicated. Biosynthesis of PHA occurs by diverse microbial species in the environment. This chapter details the wide knowledge of bacterial PHA and their significance in soil rhizosphere.

11.2

Origin of Rhizosphere

Plant roots are surrounded by regions of rhizospheres that are centered around the root and their habitat includes plant living with roots and are associated with soil where exudates deposited stimulate several microbial activities that are significantly implied in plant health and disease, and further focusing on the ecosystems terrestrial and agriculture. Microbes whose abundance and metabolic activity promotes the growth of plant, whereas other harmful microbes develop on root exudates by growing in the rhizosphere (Amaro et al. 2019). Hiltner’s findings summarize the beginning of research in rhizosphere microbiology and also provides basic and applied scientific inventions that are useful for future. Indicators such as microbial density, enzymatic activity, and root mapping are various gradients of chemicals present in spatial limits of the rhizosphere (Pernicova et al. 2019). The composition the biotic population is mostly contingent on plant species, their texture of root, location of carbon source in plants, physico-chemical properties of soil, and a variety of microbial population, amongst the other factors. Rhizospheres are extraordinarily nutrient dense regions compared to dense soil regions and the root exudates energy, molted root cells, decaying root tissue, mucilage secretion proteinaceous substances. Interactions amongst plants and microorganisms remains symbiotic and improves carbon source to the surrounding soil from root. Soil with nutrient-rich compounds support the growth of dense and diversely populated microorganisms that mainly include bacteria and fungi. Microorganisms extract nutrients from small metabolites, which are released actively or passively into the surrounding soil from the plant. These root exudates contain various metabolites, viz., amino acids, organic acids secondary plant compounds including flavonoids and terpenoids (Pakalapati et al. 2018). Microbial population utilizes these compounds for some specificity, and further individual microbial species are utilized more effectively than another species. Invertebrates such as insects, arthropods, and nematodes are considered as primary consumers and feed on live or dead materials of plants. The digestive system of these herbivorous functions as a tool for nutrients in planta and used for transporting between the roots and the soil via evacuation (Mostafa et al. 2020). They feed commonly on microbes

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present in rhizosphere, in a category that comes under primary or secondary consumers. Plant roots can contribute carbon to the rhizosphere and deliver organic metabolites from the rhizosphere (Koller et al. 2005). 200–2000% of secondary microbial metabolites efflux increased in plant-derived amino acids through root to the rhizosphere, and this approach are thought to play a representative role in the capability of plants to modify the rhizosphere (Johnston et al. 2018). Rhizospheres are nutrient-rich regions of soil that are dynamic in nature supporting dense and diverse fauna (Ciesielski et al. 2015). Despite the challenges correlated with ecological interactions present in the soil, researchers have started to understand the complex behavioral interactions taking place in the rhizosphere. Rhizosphere biology knowledge learnt by agricultural researchers have focused soil microbes on their positive and negative relationship of plants. The detailed descriptions have been represented for many rhizosphere interactions, and the ambiguous nature of the rhizosphere still remains a challenge to scientists interested in the ecology of the plants present in soil (Reddy et al. 2003).

11.3

Sources of Bacterial PHA

11.3.1 Biosynthesis of PHA Numerous microorganisms are associated with biosynthetic pathways and belong to a diverse class of polymers that have approximately hundred different types of monomers. The alternatives for petroleum-based plastic are now unearthed to be bio-based PHAs for biopolymer production as they are from renewable resources (Adeleye et al. 2020). Bacteria producing acetyl CoA converted to PHB through several biosynthetic enzymes. Several central metabolic pathways represented including glycolytic, pentose pathway, and Krebs cycle, of which PHA biosynthetic pathway reported acetyl CoA remains key element for biosynthesis of short chain length (SCL) and medium chain length (MCL) PHAs forming PHB (3-polyhydroxy butyrate). 3-ketothiolase with two molecules of acetyl CoA forms aceto-acetyl CoA (Mitra et al. 2020) (Fig. 11.1).

11.3.2 Reduction of 3-Ketothiolase by PhaB Second step of PHB biosynthesis, acetoacetyl-CoA with NADH as a cofactor by PhaB which is reduced to (R)-3-HB-CoA. Carbon of acetoacetyl-CoA, resulting in the formation of (R)-3-HB-CoA further leveling up to polymerization (Yu 2010).

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Fig. 11.1 Biosynthetic pathway of PHA

11.3.3 PHA Polymerization of PhaC PhaC is the important enzyme in PHA biosynthesis representing CoA thioesters of various HAs and polymerization. More than 150 HA CoA are required to characterize PhaC and recently only 60 different phaC genes have been denoted which defines the important role of PhaC that produces PHAs. The crystal structure of PhaC has been determined through various attempts and elucidated its catalytic mechanism over the past 30 years. However, crystallization of PhaC has so far proven unsuccessful (Kumar et al. 2019). Hydroxy butyryl-CoA polymerizes to form PHB and the propagation proceeds by additional bonding to develop PHAs representing 80% of bacterial cells. Solvent extracted PHAs bacterial cell mass grown in the presence of sugars, carbohydrates, and are focused on gene transfer pertaining to significant research activity (Mostafa et al. 2020).

11.4

Properties of PHA

PHB coming under PHA develops as good resistance materials to moisture and aroma barrier properties. High biodegradability and biocompatibility of PHAs makes it eco-friendly polymers for various applications in different fields. The most important physical and thermal property of PHA refers to molecular mass and crystallinity index (Anjum et al. 2016). Due to the chemical diversity, PHAs properties are considered similar to conventional plastics. The rate of degree of polymerization maximizes up to 30,000 that validates high molecular masses. Further consistent molecular mass and molecular weight distribution of a particular polymer represents characteristics for its commercial suitability and stability for manufacture (Costa et al. 2019). According to Chee et al. (2010), the crystallinity range of PHA between 60 and 80% referred as rigid and remains more elastic, flexible crystallinity with low degree of PHA makes it applicable for various industrial processing. Polyhydroxy butyrates are brittle and fragile with the ability to elongate less and a breaking point below 15%. PHA are generally degraded by various microbes in different environmental conditions. This results in production of

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CO2 and methane forming anaerobic digestion several years including various factors, viz., pH, temperature, humidity, microbial activity, other nutrients, and polymer properties such as crystallization and composition. PHA are highly dense and do not float on marine platforms and hence discarding these sediments are effortless. Mechanical properties depending on the monomer units exhibiting hard crystalline polyesters to be more elastic comparable to PP(Polypropylene). Addition of nanofillers blending with other polymers decreases polymer flexibility but are good barriers against organic solvents (Anjum et al. 2016; Mostafa et al. 2020). Basically, the different monomer units of PHA, viz., short chain length, medium chain length have high crystallinity and with respect to Young’s modulus, tensile strength, and oxygen permeability of PHB like isotactic PP(Polypropylene) representing excellent packaging material. Replacing synthetic plastics with these bioplastics are important for degradation problems prevailing in the environment.

11.5

Types of PHA

PHAs differ in R group and consist of 3-hydroxy fatty acids and common types are in Table 11.1. Linear chain polyesters of PHAs containing monomers of hydroxy acid (HA) that are linked to an ester bond. The ester bond is recognized by a carboxyl group that is connected to the hydroxyl group (Singh et al. 2017). Mcl-PHA are certain side chain length HA showing elastomeric properties. Different temperature conditions indicate various polymers and make them suitable for packaging films. Examples of Scl PHA are PHO (3-Polyhydroxy octanoate) and PHN (3-Polyhydroxy nonanoate). Typical examples of Mcl-PHA are 3-hydroxyhexanoate (HHx), 3-hydroxyheptanoate (HH), and 3-hydroxydecanoate (HD) are identified in the largest group of natural polyesters (Table 11.2). PHA Polyhydroxyalkanoates (PHAs) polymers are produced by several microorganisms and stores as intracellular organelles. For example, Chemolithotrophic bacteria Cupriavidus necator (Ralstonia eutropha), Cupriavidus metallidurans, and Alcaligenes latus (Suriyamongkol et al. 2007). Several Table 11.1 Classification of PHA S. No 1 2 3

Name of PHA Poly(3-hydroxybutyrate) Poly(3-hydroxyvalerate) Poly(3-hydroxyhexanoate)

R group CH3 CH2CH3 CH2CH2CH3

Short form PHB PHV PHHx

Table 11.2 Types of PHA on chain length S. No 1 2 3

Scl PHA (short chain length) 3-Polyhydroxyoctanoate (PHO) 3-Polyhydroxynanonoate (PHN)

Mcl (PHA medium chain length) 3-hydroxyhexanoate (HHx) 3- hydroxyheptanoate (HH) 3- hydroxydecanoate (HD)

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Pseudomonas sp. such as P. oleovorans and P.aeruginosa produce PHA under Gram-negative bacteria. Gram-positive bacteria producing PHAs are Bacillus sp. B. subtilis, B. thuringiensis, B. cereus, and increase PHA synthesis. Several Halophilic and Halomonas sp. based on various factors molecular weight and type of feed produce PHA.

11.6

Ecological Niche of Bacterial PHA Production

11.6.1 Hydrocarbons Hydrocarbons form effective production of PHAs approximately 30% yield of the bacterial cell weight. PHAs from hydrocarbons have propyl, dodecyl alkyl groups, and aromatic hydrocarbons develop substrates from terephthalic acid in PET. They emulsify the effective production of hydrocarbons by a number of Pseudomonas strains. Recently, PHA production from hydrocarbons are low compared to others and further improvements in production, processing has to be established for industrial scale. PHA derived from hydrocarbon in waste material showed high impact with synthetic plastics. Hydrocarbons utilized from bacterial strains such as Pseudomonas sp., R. eutropha are decomposed rapidly compared to plastic particles (Samorì et al. 2019). Other hydrocarbons in interest for PHA production are organic matter in oil fields and substrates of methane. Furthermore, soil bacteria rhizosphere shows favorable production hydrocarbons and provides a sustainable approach for the synthesis of PHA (Wongsirichot et al. 2020).

11.6.2 Halophiles Halophiles or Halophilic bacteria generally reported in oceans, salt lakes, marshy land areas, are Archaea bacteria that produce PHA. Bhardwaj et al. (2014) reported that the microbes require salt to grow and optimally 5–10% stating PHA accumulation by archaea in the Dead Sea. PHA from halophiles grow in salt conditions and about 50–60% production in its cell weight, representing an excellent carbon source. Halophiles at 3–5% salt concentration accumulate 55% PHB. Several studies show halophilic archaea bacteria such as Halococcus sp. and Halorubrum sp., independent of nutrient conditions develop PHB. Halophiles produce PHA for synthesis of polymers at industrial level. Halotolerance bacteria are from moderate to extreme from 20 to 30% salt concentration. Use of halophiles in processing of PHA makes it a low-cost substrate and more efficiently produced from the bacterial cells and further develops biocatalysts for industrial PHA production (Sanhueza et al. 2019). Researches described show excellent potential of halophiles for production of PHA via biotechnological techniques for various industrial applications.

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11.6.3 Photosynthetic Bacteria Cyanobacteria are represented as photosynthetic, and they produce PHA during oxygenic photosynthesis that have natural sites to store PHA. Species-specific bacteria producing PHB reports the presence of PHA in cyanobacteria (Kai and Loh 2013). Studies reported production of PHA by cyanobacteria and their capability to produce PHB sunlight energy which reduces the production of greenhouse gases (Gasser et al. 2009).

11.6.4 Antibiotic Factors Rhizosphere are untapped reservoirs of PHA, and some microbes are Burkholderia terricola, Lysobacter gummosus, and Pseudomonas extremaustralis produce PHA. Azospirillum brasilense is a growth-promoting rhizobacterium that has been reported to produce PHA. Antibiotic factors are reserved in the roots of plants that impulse hydroxyalkanoates production, and these microbes include the ability to adapt environmental conditions to promote growth of rhizosphere habitat (Obruca et al. 2018). Azospirillum brasilense synthesizes nitric oxide, carotenoids, and other nutrients further utilizing PHA polymers for shelf life of bacteria. Mostly, production of soil inoculants, biofertilizers containing beneficial bacteria for agricultural purposes. Molecular techniques and cultivation patterns characterize PHA production from rhizopheres. PHA producers by the fact the rhizosphere roots contain ample sources of inorganic and carbon nutrients are in limited quantity. PHB production is found in different rhizobia established in Sinorhizobium meliloti within alfalfa. PHA stored in roots helps in rapid colonization, efficient nodulation, and nitrogen fixation (Volova et al. 2019). PHB production plays an important role in antibiotic factors thereby imposing nitrogen supplementation.

11.7

Bacterial PHA in Rhizosphere

11.7.1 Screening of PHA from Bacterial Sources PHA produced from microbes provide unbalanced growth conditions, and this happens when nutrients such as nitrogen, phosphorus, or sulfate are in limited quantity. PHA and PHB are present in numerous microorganisms. Bacteria such as Acetobacter, Bacillus, Archaebacteria, Methyl bacteria, and Pseudomonas have been found to synthesize PHA. To isolate PHA-accumulating bacteria from diverse sources, several researches have been carried out and different strains were selected efficiently. Industrial effluents, dairy waste, domestic sewage, and activated sludges are found to be the prime sources of active PHA. Higher level PHA producers from tannery effluent and sewage sludge and further isolating PHA-accumulating bacteria from nature, it is necessary to screen collection of bacteria through molecular techniques. Stains specific to PHA are detected viable colony staining technique

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and the ability of the isolates was further studied by Sudan Black staining method (Sujatha et al. 2005). Food waste, paper mill waste, molasses, cane sugar waste, and oil plant effluents are sources of PHA, and other agricultural wastes also provide PHA-producing microorganisms.

11.7.2 Characterization and Identification of PHA from Bacterial Sources Isolates of PHA from bacterial sources were screened under Sudan Black staining for 48 h incubation on E2 medium and analyzed for Gram type of isolated bacterial colonies. Identification of the bacterial colonies are further characterized by biochemical tests. PHA are a range of Gram-positive and Gram-negative bacteria, mostly bacillus species. Several researchers reported PHA production from different Bacillus species. Singh Saharan et al. (2014) reported PHA production from B. subtilis, B. megaterium, and characterization from industrial wastes. Pseudomonas species have also been studied for formation of PHA by Pseudomonas oleovorans. Pseudomonas sp. isolated from large quantities industrial waste showed polyhydroxy butyrate (PHB) (Ayub et al. 2009). Fermented molasses mash from sugar industry waste represents potential carbon sources and corn oil also showed high PHA content. The bacterial colonies are developed around 48–72 h to screen PHA development with axenic strains nature (Ayub et al. 2007). DNA extraction and characterization from cultures developed using manufacturer’s instructions manual of the DNeasy Blood and Tissue kit (QIAGEN GmbH, Hilden, Germany) that showed uniform colonies and cellular morphology which were assessed spectrophotometer (Suriyamongkol et al. 2007). Further characterization of these bacterial PHA were processed through PCR amplifications of the 16S rRNA and phaC genes with general procedures and effective bacteria-producing PHA are identified. The identification and characterization of the bacterial PHA in the rhizosphere was reported by Gasser et al. (2009) depicting Pha genes stress resistance associated with PHA and PHB accumulation. Changing environmental conditions and other features are responsible for production and biosynthesis.

11.7.3 PHA Production from Bacterial Sources High quantities of PHA-producing bacteria present in marine, soil, and effluents has been done screening using culture-specific approaches. Gram-positive and Gramnegative bacteria synthesize various PHA. PHA synthases are key enzymes degraded by phaC gene that polymerize PHA monomers (Tarrahi et al. 2020). PHAs of short chain length contain enzymes of only one type (PhaC), and medium chain length PHAs (mcl-PHA) contain class II and polymerize showing different rheological properties. Desirable biotechnological developments, cultureindependent methods for the detection potential of PHA producers are showcased and some bacteria occupy more than one phaC Cupriavidus necator. Evidence for

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PHA production in Alpha proteobacteria and Gamma proteobacteria in hypersaline environments characterized by metabolic diversity (Roja et al. 2019). In situ analysis of PHA and its monomer’s are studied through biotechnology that can be implemented on an industrial scale for various applications in food, pharmaceutical, and cosmetic agents. Within the last few decades, the main contributors of high cost production in PHA are highly pure substrates that account for 45% in total cost of production. Furthermore, renewable sources are explored by researchers and they develop bioprocesses for by-product utilization. By-product are important for PHA production.

11.8

Factors Affecting PHA Production

Nutrient limitation is the primary factor affecting PHA production that is directed towards increased biomass growth. Phosphorus and nitrogen in low concentration provide limited conditions for PHB synthesis. Bacteria removing substances like phosphorus was obtained from waste and are able to produce PHB. Optimizing the conditions for PHA production focuses factors like oxygen or nitrate as electron acceptor, nitrogen concentration (NH4Cl), phosphorus (KH2PO4), and carbon concentration (acetate). The carbon/nitrogen (C/N) ratio for PHA production is 28.3 that reflects 1.8 folds increase during PHB production. Activated sludge also a source of PHA was found to be effective. Box-Behnken design requires optimization of the cultural conditions that maximizes PHB production and parameters for cultivation implies ammonium sulfate as nitrogen source, glucose as carbon source, KH2PO4, and Na2HPO4 as phosphorus source that provides maximum production. PHA production is prominent by the addition of various acids including citrate (0.5%) and acetate (0.5%) production from Aulosira fertilissima, resulting to form PHB. Further, mixture of acetic acid and butyric acid represented high productivity of PHB (Lee et al. 2019). PHA polymer composition acidogenic effluents (AE) varied and PHA derived from synthetic acids were found to be 91%, and AE were found to be 84%. Pseudomonas putida and Pseudomonas aeruginosa were studied and reported to specify the route of PHA metabolic biosynthesis (Singh Saharan et al. 2014). Composition of monomers producing PHA demands challenging in research field for its production polymers and high substrate availability of PHB results for faster PHB production (Wong et al. 2020). pH plays a vital role in PHA productivity and its composition of monomer. Study on effect of pH on fermentation with an alkaline pH of 9 showed high production level of PHAs. pH range also affected composition of polymer and PHA accumulated pH 8.5–9.5 increases PHA production. Microorganisms include cytoplasmic pH compatible with functioning of cell that responds to change in pH of the external environment. PHA production increased at pH of 5.5–9.5 on an account of 10–30% mol (Velázquez-Sánchez et al. 2020).

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Applications of PHA

Microbial PHAs have considerable potential towards various fields including pharmaceuticals, agriculture, regulated for pesticides, manufacturing biofuel, and medicine in surgical substitutes. PHA as nanocomposites are utilized in bioengineering. The biopolymer plays a vital role with biocompatibility, bioavailability, and biodegradability properties. PHA-based nanocomposites are utilized for cell proliferation in tissue engineering. Biodegradable, eco-friendly agricultural products, viz., fertilizers, insecticides, and pesticides for protecting the plants and saplings. PHAs are biodegradable polyesters offering greater solution in waste management and present as substitution for conventional plastic. Nanocomposites from PHA are also used in developing antibacterial applications. For example, ZnO-based nanocomposites are good disinfectants preventing bacterial proliferation in food packaging and medical instruments. PHA in enzyme immobilization highly acknowledged application to provide impact on biomaterials. Enzyme immobilization is a familiar field for purification and nanoencapsulation of active materials from preventing degradation through various factors. Nanoprecipitation technique to synthesize PHA from dairy waste developed and performed active production PHA from microorganisms (Zhang et al. 2018). Drug delivery of nanoparticles is ideal to implement delivery of bioactive compounds reducing drug degradation and deactivation. PHA-based nanoparticles are biodegradable and finally optimize the patient compatibility as drug delivery systems should be removed in the body to prevent potent toxicity. These polymers are nonspecific esterase and lipases that are widely incorporated for hydrophobic rugs and proteins (Chanprateep 2010). PHAs are less toxic when compared with synthetic polymers and are designated as promising sources for therapeutic applications. Protein purification has been a crucial process. PHA nanoparticles play a major role in protein purification. PHAs are used in the food industry for various applications, viz., antimicrobial films, thermal stability, provide strength and toughness for food packaging material (Liu et al. 2020). Polyhydroxyalkanoates (PHAs) are highly biodegradable and processing versatility to replace fossil fuel-based plastics. They generally originated from microbial cultures possessing main properties as water insolubility and resistance to ultraviolet rays. Further, biocompatibility and degradability are anaerobically compared to other polymers. PHB degradation takes place with production of polymers with short chain length and PHA made by extrusion for developing flexible plastics molded to various shapes (Tarrahi et al. 2020). PHAs can be applied in a variety of applications including wall paint coatings, textile materials, films for food packaging, medicinal products, farming agriculture application, for commercial availability. PHA can be used as bioplastics in cables, connectors, and in electronics manufacturing companies, viz., Nokia, Samsung, Sony, Fujitsu, for their goods.

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The premium cost of production develops support for green products and consumer interests. PHA biopolymers have a flexible nature that provides extension to produce materials such as caps, bottles, blister packs, and other containers for various customer goods industries. Represents as a promising polymer with properties implying barrier and mechanical strength compared to other bioplastics such as polylactic acid (Adeleye et al. 2020). PHA polymer provides advanced mechanical strength making the packaging foils suitable for packaging food products. Further, possess basic property such as excellent oxygen transmission rate (OTR) and water vapor transmission rate (WVTR) representing the primary role of any packaging material that makes it a potential choice for food packaging (Costa et al. 2019). Flexibility of PHAs shows promising property for various applications in agriculture, and biodegradable plastic requires release mechanism showed control characteristics and can be implemented in fertilizer. PHA pellets are developed technologies that are placed on fields of the soil showing gradual degradation over a long period further reduce the use of fertilizer and cost of labor. Industrial scale production of PHA is in focus by engineering biocatalysts via synthetic biology tools. Industrial biotechnology focuses the effort to minimize PHA production cost mainly by engineering strains of higher PHA production and thereby provide less energy consumption, for high value-added applications during PHA production,

11.10 Future Prospects and Challenges PHA represents a high potency of low-cost biodegradable plastics, and there are still some disadvantages for the consumption focusing on production. Nonbiodegradable plastics and other problems influencing production are PHA properties. Economic use of waste feedstock from microbial cultures leads to the production of PHAs and provides greater sustainability. Ongoing research on different methods for the improvement of the yield of PHA are done using genetic modification of the bacteria that are isolated from waste materials. Diverse polymers available in industrial applications with high value functionalities should be focused for improved PHA production. Further investigations were made to reduce the cost of production of PHA polymers and have an impact on several researchers for developing environmentally friendly polymers. Despite the significant advances in PHA production and accumulation research towards molecular advances enhance the survivability of bacteria. Advances in this area in future could benefit the industry of bacterial inoculants and the capabilities of the microorganisms to establish the target niche is of utmost importance. Advanced technology requires upgradation to develop an integrated system for microbially synthesized polymers from soil bacteria rhizosphere. PHAs produced from various sources, effluents, and wastes developed using similar microorganisms would help to reduce environmental pollution and hence recommended for commercialization.

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Acknowledgments One of the authors SB, thankfully acknowledges Tamil Nadu Veterinary and Animal Sciences University for providing the necessary facilities to carry out the work.

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Role of Microbial Biofilms in Agriculture: Perspectives on Plant and Soil Health

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Anupam Gogoi, Mandeep Poudel, Jagajjit Sahu, and Geetanjali Baruah

Abstract

Microbial communities, especially beneficial microbes are a boon to plant health as well as mankind. Regardless of the works that have been done in this field, still, a long way is ahead of the researchers to employ notable microbes in the benefit of plants to fight against various stresses and diseases. Biofilms are the multicellular assemblies or aggregates of differentiated microbial communities embedded in an extracellular matrix which have proven to be an efficient tool for the sustainable management of crops against multiple stresses. This chapter aims to provide deep insight into the biofilm-producing microbiota and their roles in crop protection. The knowledge garnered from the published work led us to information regarding the functional attributes of various biofilm-producing microbiota. As the literature suggested maximum numbers of biofilms used are from the bacterial community such as Azospirillum brasilense, Bacillus amyloliquefaciens, and Pseudomonas fluorescens. The biofilms are beneficial in enhanced nutrient solubilization, nitrogen fixation, sequestering, enhancement of plant biomass, and many other important physiological processes. Besides, we also attempted a basic scientometrics approach to provide the most frequent keywords for the published articles for various types of microbes forming biofilms. The prospects of biofilm

A. Gogoi · M. Poudel Department of Plant Sciences, Faculty of Biosciences (BIOVIT), Norwegian University of Life Sciences (NMBU), Ås, Norway J. Sahu National Center for Cell Sciences (NCCS), University of Pune Campus, University Road, Ganeshkhind, Pune, Maharashtra, India G. Baruah (*) Environment Division, Assam Science Technology and Environment Council, Guwahati, Assam, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_12

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in crop protection depend upon a better understanding and may be combined with multi-omics approaches. This chapter establishes the efficiency of the use of biofilms disclosing sustainable ways of fighting against stress conditions and maintaining plant health. Keywords

Beneficial microbe · Sustainable agriculture · Microbiota · EPS · Soil restoration · Quorum sensing

12.1

Introduction

Plants encounter diverse groups of microbial communities such as beneficial, pathogenic and commensals, and abiotic stresses such as drought, salinity, heat-stress, pH, and temperature in naturally growing environments (Knief et al. 2011; Narsai et al. 2013; Ahmad et al. 2019). Due to the ever-increasing world population and constant global climatic changes (Cleland 2013; Ekwurzel et al. 2017), threats to food security and sustainability have become a growing concern. Meanwhile, a combination of environmental stresses and deterioration of soil health due to excessive use of synthetic fertilizers and pesticides hampered the whole agricultural scenario. It is estimated that disease caused by biotic factors account for 20–40% of annual crop losses worldwide (Oerke and Dehne 2004; Savary et al. 2012). The abiotic stresses also contribute significantly to the reduction of crop yield and productivity (Ciais et al. 2005; Francini and Sebastiani 2019). Several management tactics including the use of pesticides, synthetic fertilizers in combination with resistance breeding, and agronomic practices have been employed in recent decades to overcome the environmental stresses. However, the continuous application of synthetic chemicals not only deteriorates soil health but also imparts residual toxicity in foods (Aktar et al. 2009; Bourguet and Guillemaud 2016). The rapid evolution of pathogens to overcome plant resistance genes and pesticides limit their long-term use in the field (McDonald and Stukenbrock 2016). Thus, an alternative, efficient, and sustainable approach is needed to tackle the present problems associated with plant and soil health. Most soil-borne pathogenic microbes in the cropping environment are often counterbalanced by the metabolic activities of associated beneficial microbiome (Wei et al. 2019). These beneficial microbes have been a subject of research and have drawn attention towards sustainable management of crops against environmental stresses. Microbial cells physically interact with plant surfaces via the formation of multicellular assemblies known as biofilms (Danhorn and Fuqua 2007). Biofilms are self-assembled, aggregates of microbial cells or a coherent mixture of differentiated microbial community embedded in an extracellular matrix of polymeric substances that are associated with an inert or biotic surface (Stoodley et al. 2002; Danhorn and Fuqua 2007; Vlamakis et al. 2013; Velmourougane et al. 2017). Cells adhere to surfaces via extracellular polymeric substances (EPS), which is a

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complex mixture of exopolysaccharides, lipopolysaccharides, proteins, and DNA (Whitchurch et al. 2002). Biofilm formation and maturation is mediated by EPS production and triggered by environmental and quorum sensing signals (Flemming et al. 2016). Since the production of these biomolecules is energy driven, their functions should carry advantage to the producer microbial community. Most of the benefits of biofilms are attributed to environmental protection such as shielding from antimicrobial compounds, drought, and heavy metals (Wolfaardt et al. 1999; Sandhya and Ali 2015; Flemming et al. 2016; Zainab et al. 2020). Slime formed by EPS helps in sequestering carbon, water, nutrients, and facilitated cell–cell communication within the microbial community and with plant cells (Moons et al. 2006; Islam et al. 2016). Studies on EPS extracellular DNA revealed that the presence of DNA molecules can be a template for genetic exchange, signaling, and provide structural integrity (Flemming et al. 2016). Biofilm formation is an inherent mechanism adapted by many microbes including bacteria, fungi, algae, and archaea, irrespective of their beneficial or pathogenic traits to their hosts (Velmourougane et al. 2017). Beneficial biofilm-producing microbes are a subject of interest for agricultural and industrial applications. Several microbial species including bacteria such as Bacillus, Pseudomonas, Rhizobium, Azospirillum, Azotobacter, and Burkholderia; cyanobacteria—Anabaena and Nostoc; filamentous fungi— Trichoderma promotes plant growth and soil health. These beneficial plant microbiomes help solubilizing and up taking of macro- and micronutrients such as Phosphorus (P), Nitrogen (N), Potassium (K), Magnesium (Mg), Zinc (Z), Copper (Cu), and Iron (Fe) by their hosts (Al-Nahidh and Gomah 1991; Ma et al. 2009; Wang et al. 2020). They also protect crops from various biotic and abiotic stress including drought, low/high temperature, change in soil pH, and salt tolerance. The slime layer produced by the EPS matrix act like a glue that helps in soil aggregation and thus improves soil structure, health, and fertility (Chenu and Cosentino 2011; Costa et al. 2018). In this chapter, we addressed the roles of microbial biofilms in various aspects of stress management in plants and how they promote soil health and sustainability in agriculture. We have also tried to shed light on the publication patterns and research outputs in this field via a basic scientometrics analysis to help young researchers get clarity in this regard.

12.2

Biofilm-Producing Microbes Categorically with Special Emphasis on Agriculturally Important Microbes (AIMs)

Plant and soil-associated microbial communities impart several traits either beneficial or pathogenic to their hosts and their growing environments. Most microbes form biofilms to interact with their ancestral population via a series of developmental stages. Stoodley et al. described five biofilm developmental stages as (1) primary attachment of cells to a physical surface, (2) formation of EPS to firmly adhere cells together, (3) cell differentiation and biofilm development, (4) maturation of biofilm architecture and the slime-layer formation, and (5) disassembly and dispersal of individual cells from the differentiated biofilm (Stoodley et al. 2002). Cell–cell

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communication during the differentiation process involves signaling mechanisms that control the phenotypes of microbial communities. In bacteria, these mechanisms include the exchange of organic molecules, proteins, or transmission of electrical signals by ion channels (Prindle et al. 2015). Studies on the role of chemical signaling (also known as quorum sensing, QS) that involve several diffusible sensing molecules have been a subject of intensive research. These include acyl-homoserine lactones (AHLs), autoinducing peptides (AIPs), γ -butyrolactones. 3,4-dihydroxy-2heptylquinoline (PQS), 3OH palmitic acid methyl ester, and cyclic dipeptides, commonly known as autoinducer (reviewed by Waters and Bassler 2005). QS is a signaling process initiated by specific diffusible sensing molecules that carry intercellular chemical signals which result in an inducible gene expression at a population level to adapt to the local environmental changes. The knowledge of QS is not only limited to bacteria but has been extended for closely related cyanobacteria (Sharif et al. 2008) and other organisms including filamentous fungi (Padder et al. 2018), algae (Zhou et al. 2016), oomycetes (Kong et al. 2010a, b), and archaea (Rajput and Kumar 2017; Kaur et al. 2018). Some bacteriophage can also bind to bacterial QS molecules via their genome encoded LuxR-type receptor (Silpe and Bassler 2019) or can sense the metabolic state of host cyclic-30 ,50 -AMP (cAMP) receptor protein (CRP) to determine host population density (Laganenka et al. 2019). An increase in bacterial population modulates the transition of virus particles from lysogenic to lytic state (Laganenka et al. 2019).

12.3

Roles of Microbial Biofilm in Crop Protection

12.3.1 Disease and Pest Resistance M Biofilm-producing beneficial microbes from different taxa including bacteria, fungi, algae, and archaea play important roles in shaping plant growth and development parameters and protect crops against various biotic and abiotic stresses (Ariosa et al. 2004; Ribaudo et al. 2006; Sun et al. 2010; Fontenelle et al. 2011; Song et al. 2019). These microbes colonize in the rhizoplane (Hansen et al. 1997; Benizri et al. 2001), or in the outermost zone of the rhizosphere, i.e., in the soil compartments called as ectorhizosphere (Whipps 2001; Lopes et al. 2016), or inside plant tissues such as root hair, cortex, and vascular bundles to establish a mutualist interaction with their host and its environment (Hallmann 2001; Compant et al. 2010; Jia et al. 2016; Liu et al. 2017). Colonization of beneficial microbes within the plant intercellular spaces are known as endophytes. Endophytic cohabitation and rhizosphere inhabiting microbes enhance plant immunity mechanisms, thereby primed the host to combat subsequent attack by pathogenic microbes and pests (Van Wees et al. 2008). Plant tissues colonized by a biofilm often generate mobile signals that trigger induced systemic resistance (ISR) in the whole plant. For instance, root inoculation of Paenibacillus polymyxa MAS100 in potato (Solanum tuberosum) suppressed growth of the bacterial wilt pathogen Ralstonia solanacearum and reduced disease incidence up to 20% after inoculation of the pathogen (Soliman 2020). Besides, the

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rhizobacteria Paenibacillus spp. could potentially be used as a biocontrol agent due to its dual role as an antifungal and nematicide against Fusarium wilt and root gall disease caused by Meloidogyne incognita, respectively (Son et al. 2009). Seed inoculation of two endophytic bacteria Bacillus pumilus and B. amyloliquefaciens in tomato reduced disease severity of up to 62% against bacterial speck disease (Lanna-Filho et al. 2017). Several studies have reported that these bacterial species induced expression of several pathogenesis-related protein-coding genes including PR1, PR2 and PR3, and other defense-related enzymes to reduce viral load and multiplication of many viruses including Bean common mosaic virus, Cucumber mosaic virus, Papaya ringspot virus, Pepper mild mottle virus, Tobacco streak virus, Tomato chlorotic spot virus, Tomato mottle virus, and Tomato yellow leaf curl virus in many crop species (Murphy et al. 2000; Ahn et al. 2002; Jetiyanon and Kloepper 2002; Udaya Shankar et al. 2009; Abdalla et al. 2017; Vinodkumar et al. 2018; Guo et al. 2019). Many other bacterial species including P. fluorescens, P. putida, B. subtilis, B. cereus, B. sphaericus, and related species induce ISR to protect crops against many viral diseases (Beris and Vassilakos 2020). Some fungal species including Aspergillus sp. strain NBP-08, Penicillium chrysogenum, Penicillium oxalicum, Penicillium sp. strain NBP-45, Talaromyces sp. (isolates NBP-61, NBP-66 and NBP-67), Trichoderma harzianum, and many others can also contribute in plant immunity by modulating hormonal signaling pathways and thereby the expression of defense-related genes and gene products such as β- 1,3-glucanase, peroxidase, chitinase, lipoxygenase, polyphenol oxidase, and superoxide dismutase (Chowdappa et al. 2013; Murali et al. 2013; Murali and Amruthesh 2015). These antimicrobial proteins in plant functions as inhibitory enzymes by blocking pathogen growth via disintegration of cell wall through hydrolytic cleavage of β glycosidic bonds, polysaccharide cross-linking and lignification in plant tissues, facilitate the accumulation of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide (O2) that are toxic to the invading pathogen (Sudisha et al. 2012). The role of hormonal pathways including salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and auxin (Aux) signaling in plant defense against pathogens, and pests have been well documented and reviewed (Pieterse et al. 2014). Regulation and crosstalk between phytohormonal signaling pathways are key mechanisms of ISR to biotrophic and necrotrophic pathogens (Glazebrook 2005). Increased expression of phenylalanine ammonia lyase (PAL), a central regulator of the SA biosynthetic pathway has a clear link to resistance against most biotrophs, whereas JA/ET hormonal pathways are more active against necrotrophic pathogens. Nevertheless, five rhizospheric plant growth-promoting fungi (PGPF) antagonistic to the necrotrophic fungi Colletotrichum capsici showed induced PAL enzyme activity in Chilli (Capsicum annum L.) (Naziya et al. 2020). Plant hormones also play protective roles against insect herbivores and are triggered by beneficial microbes. For instance, ISR triggered by root-colonizing microbial biofilms increased expression of JA-dependent gene—Lipoxygenase 2 (LOX2) and JA/ET-dependent genes— Plant Defensin 1.2 (PDF1.2) and Hevein-like protein (HEL) in Arabidopsis and thereby primed host against leaf chewing and phloem-feeding insect pests (Van Oosten et al. 2008; Pineda et al. 2012; Pangesti et al. 2015). Although, most

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microbially induced resistance against plant-feeding insects are JA-dependent, while JA-independent mechanisms via phenylpropanoid and terpenoid biosynthetic pathways are no exception (Valenzuela-Soto et al. 2010; Zebelo et al. 2016; Sharma et al. 2017). Thus, different microbial species induce ISR via various phytohormone signaling pathways, and a conserved link to describe its role against biotrophy, necrotrophy, and herbivorous pests are poorly understood. Cell wall lignification and callose deposition are two important signature mechanisms of defending priming in plants. The facilitated accumulation of these metabolites by microbial biofilms helps in strengthening of the cell wall and acts as a physical barrier for pathogen invasion and insect herbivory (Pieterse et al. 2014; Harun-Or-Rashid and Chung 2017). Several bacterial species produce volatile organic compounds or facilitate the release of herbivore-induced plant volatiles that collectively act as insect repellents, toxic to herbivores, or attract predators to feed parasitize herbivores (Ryu et al. 2004; Zebelo et al. 2016; Aartsma et al. 2017). These secondary metabolites including 2,3-butanediol, acetoin, gossypol, and other volatile compounds are air diffusible that systematically primed other parts of the same plant so that they become prepared to resist subsequent attack by pests. Thus, biofilm-producing microbes trigger ISR via various mechanisms at different levels of resistance to protect plants against various biotic stresses caused by bacteria, fungi, viruses, and insect pests.

12.3.2 Protection from Abiotic Stress In agriculture, apart from the biotic stresses, increasing exposure to various abiotic stresses including soil salinity, temperature fluctuations (low and high temperature), drought and waterlogging, mineral deficiency, and heavy metal toxicity are hostile to plant growth and development processes (Khan et al. 2014; He et al. 2018). These multilevel abiotic stress factors negatively affecting the global food production and thereby, socio-economic outcomes (Ghassemi et al. 1995; Ashraf et al. 2012). It has been estimated that abiotic factors globally contribute more to yield loss (kg/hectare) than biotic factors in major crops including corn, wheat, soybean, millet, oats, and barley (Bayer Crop Science 2008). For instance, saline stress, one of the most predominate, that cover 10% of the land surface (950 million ha; Mha) and 50% of arable land (230 Mha) globally (Carrow and Duncan 1998), is reducing the use of agricultural land by 1–2% every year (Islam et al. 2016). High soil salinity impinges various stages of plant physiological processes, viz. seed germination, seedling establishment, growth, and fertility, resulting from induced osmotic imbalance across the plasma membrane, ionic toxicity, the release of ROS, and nutrients unavailability (He et al. 2018). An interlinked of this is drought stress resulting from low rainfall that can be aggravated by a change in global temperature, high intensity of light, soil structure, and reduced water retention capacity (Salehi-Lisar and Bakhshayeshan-Agdam 2016). Drought stress is multidimensional as it affects the acquisition of plant nutrients and water, and the rate of transcription that result in reduced biomass production and crop productivity. Also, metal contamination in

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soils resulting from metal mining, sewage sludge, waste disposal, and irregular agricultural practices (application of fertilizers and pesticides) has become a serious concern as it affects about 235 Mha of agricultural land. The heavy metal contaminants (e.g., Al, Cd, Cu, Cr, Ni, Pb, and Hg) not only affect crop yields but may enhance residual toxicity in foods as it can translocate to the edible plant parts (Hart et al. 2006; Khan et al. 2014). Thus, abiotic stresses influence various parameters of plant health and affect agro-ecosystems and may pose serious threats to food security, human and animal health. The employment of microbial biostimulants is an eco-friendly and sustainable approach for effective management of environmental stresses and in promoting plant health. These microbial biofilms perform multiple beneficial roles that include enhanced tolerance to salinity and drought stress (Alami et al. 2000; Gusain et al. 2014; Islam et al. 2016), nutrient availability (e.g., N, P, K, Mg) (Chowdappa et al. 2013; Islam et al. 2016), heavy metal tolerance and ROS scavenging (Faisal and Hasnain 2005; Islam et al. 2016; Crossay et al. 2020). The mechanisms of alleviating salt stress by the plant-associated microbes include nullifying hormonal imbalance by secreting indole acetic acid (IAA) to improve plant growth parameters and counterbalance the release of stress hormone abscisic acid (ABA). During saline stress, excessive Na+ ions can induce blockage or reduce the efficiency of transporters such as high-affinity K+ transporter 1 (HKT1) and proton pump ATPase that control Na+ uptake. Beneficial microbes such as Pseudomonas induce expression of these ion transporters, thereby help plants to accumulate more K+, Mg2+, and Ca2+ to induce plant growth and to avoid ionic imbalance caused during salt stress (Yang et al. 2009; Yao et al. 2010). Previous studies have revealed that plantassociated rhizobacteria Paenibacillus polymyxa enhance transcript abundance of EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15), a drought stressresponsive gene in Arabidopsis and Achromobacter piechaudii ARV8 produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase to lower the drought stresselevated ethylene levels and restored normal plant growth (Timmusk and Wagner 1999; Mayak et al. 2004).

12.4

Role in Soil Health

Microbial biofilms can not only promote plant health but also maintain soil architecture and improve water retention capacity by forming soil aggregates (Alami et al. 2000; Sandhya et al. 2009; Deka et al. 2019), nutrient composition, nutrient solubilization, and availability (Lee et al. 2008; Wani and Khan 2010; Malviya et al. 2011; Wang et al. 2014; Pérez-De-Luque et al. 2017), carbon sequestration, nitrogen fixation, and soil fertility enhancement (Hafeez et al. 2000; Rinaudi and Giordano 2010; Prasanna et al. 2016). With increasing concern of heavy metal contamination in arable soils, bioaugmentation—“an application of cultured microbial biomass on the soil surface to remove or degrade specific contaminants from the soil” could serve as an effective strategy for improving metal toxicity. For instance, co-inoculation of sunflower (Helianthus annuus) with bacteria Pseudomonas

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libanensis strain TR1 and arbuscular mycorrhizal fungus (AMF) Claroideoglomus claroideum strain BEG210 increased plant biomass and promote phytoremediation of nickel (Ni) from contaminated saline soil. Increased production of ACC deaminase, IAA, and siderophore by these microbes might help in efficient uptake of Ni2+ ions from the soil (Ma et al. 2019). An important attribute of soil health is soil structure while influencing both the nature and content of organic matter. The basic units of soil structure are soil aggregates, which determine the physical and mechanical properties of soil such as porosity, hydraulic conductivity, water retention capacity, aeration, and temperature. Plant mucilage and microbial EPS play important role in binding soil units together and increase organic matter content (Watt et al. 1993). Several studies have shown that the application of microbial EPS resulted in stable soil aggregation and substantially increased nutrient uptake by plants. For instance, root adhering and EPS-producing Rhizobium spp. strain YAS34 significantly increased soil macropore volume of up to 12–60 μm in diameter after inoculation in soil and increased sunflower shoot and root dry mass up to 50 and 70%, respectively (Alami et al. 2000). A similar effect was observed in wheat inoculated with Paenibacillus polymyxa CF43 that increased the ratio of root-adhering soil dry mass to root tissue dry mass by 30–100% in treated soil (Bezzate et al. 2000). Microbial biofilms could also play an important role in maintaining carbon (C) cycling via sequestration of atmospheric CO2 in soil, providing the potential for large-scale C sinks. Thus, it can aid in the possible management of leading global problems such as global warming and soil conservation.

12.5

Impact on Plant Growth

Plant growth parameters are essential features for monitoring crop yield and productivity. With diminishing natural resources, it is important to focus on different growth parameters for greater yield with fewer inputs. On the other hand, increasing focus on plant biomass and intensifying food production by sustainable means are among the top 12 areas of the 2030 agenda of the Food and Agricultural Organization (FAO) to end poverty and hunger. It has been estimated that food production must rise to 60% to feed the global population that is projected to reach 10 billion by 2050 (FAO 2009). An overall increment for food production by sustainable means is a challenging goal for the current agricultural sector. In this case, a balance employment of microbial communities to enhance plant growth parameters could potentially create a new avenue for sustainable agriculture. The use of microbes to enhance plant growth and biomass has been well documented in many crop species (Ribaudo et al. 2006; Lee et al. 2008; Canellas et al. 2013). These include Amaranth (Amaranthus gangeticus), Arugula (Eruca sativa), Capsicum (Capsicum annuum), Chickpea (Cicer arietinum), Chinese cabbage (Brassica rapa subsp. chinensis), Mustard (Brassica campestris), Chrysanthemum (Chrysanthemum morifolium), Cotton (Gossypium barbadense and Gossypium hirsutum), Cucumber (Cucumis sativus), Groundnut (Arachis hypogaea), Lettuce

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(Lactuca sativa), Maize (Zea mays), Mungbean (Vigna radiata), Red sage (Salvia miltiorrhiza), Ribbed bog moss (Aulacomnium palustre), Rice (Oryza sativa), Sesban (Sesbania sesban), Soybean (Glycine max), Strawberry (Fragaria. spp. and F. ananassa), Sunflower (Helianthus annuus), Tea (Camellia sinensis), Tomato (Lycopersicon esculentum and Solanum lycopersicum), and Wheat (Triticum aestivum). A detailed list of microbial communities involved in plant growth and development, and their role in defense against biotic and abiotic stresses, and promotion of soil health is documented in Table 12.1. In the interest to access, the connections between different species producing biofilms and the crop plants that have been benefited, a chord diagram was created using Circos (Krzywinski et al. 2009) (Fig. 12.1). The input for the Circos tool was created with the help of in-house R scripts from Table 12.1. As the figure mentions, the Azospirillum brasilense, Bacillus amyloliquefaciens, and Pseudomonas fluorescens were the three most connected biofilm-producing microbiota. All the three species were bacteria with every six connections to six different crop species. In the case of crop species, both Tomato and Rice were found to be connected to a maximum of ten species producing biofilms. Most of the functional attributes influenced by beneficial microbes include enhanced nutrient solubilization and uptake, fixation of atmospheric nitrogen by nodules formation, sequestering iron via the production of siderophores. These actions positively affect plant growth parameters such as (1) increase in root and shoot length, (2) seed germination, and seedling vigor, (3) increase in grain production, and overall plant biomass. Change in the level of two phytohormones—IAA and ethylene both related to plant growth is influenced by microbial biofilms. Most studied Rhizobium and Azospirillum species produce IAA that facilitate nodule formation in the host plant (Ribaudo et al. 2006; Leggett et al. 2017). Similarly, mutants of soybean Bradyrhizobium elkanii deficient in IAA production induced fewer root nodules than the wildtype strain (Fukuhara et al. 1994). In contrary to IAA, ethylene can affect plant growth parameters in different ways such as inhibiting root elongation, promoting fruit ripening and senescence, and inhibiting nodule formation. Many plant growth-promoting bacteria such as Rhizobium leguminosarum, Pseudomonas spp., Azospirillum spp., and Burkholderia unamae synthesize the enzyme ACC deaminase, which converts ACC (aprecursor of ethylene biosynthesis) into ammonia and α-ketobutyrate (Glick et al. 2007). As a result, the level of ethylene decreases, and therefore the plant growth inhibitory role of the hormone could be overcome (Onofre-Lemus et al. 2009; Glick 2012). Thus, these microbes modulate phytohormone pathways to influence plant growth parameters and enhance plant biomass.

12.6

Factors Affecting Biofilm Formation

There is a lot of information is yet to unveil contributing to the current knowledge on how plants regulate the microbial association. The process of biofilm formation is dependent on multiple factors. Some of such key factors are mentioned below.

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Table 12.1 Examples of biofilm-producing microbiota from different taxa involve in promoting plant and soil health Microbes Bacteria Azospirillum brasilense Azospirillum lipoferum

Azospirillum brasilense Bacillus xiamenensis

Bacillus cereus

Bacillus subtilis Bacillus amyloliquefaciens Bacillus subtilis strain QST71

Functional attributes

Crop plant

References

Enhanced production of phytohormones, enhanced root and shoot length, increased root hairs and side roots

Capsicum annuum Gossypium barbadense Gossypium hirsutum Lycopersicon esculentum Triticum aestivum Oryza sativa

Ribaudo et al. (2006), Bashan (1998)

Sesbania sesban

Din et al. (2020)

Vigna radiata

Islam et al. (2016)

Arabidopsis thaliana

Ryu et al. (2004)

Cucumis sativus

Garcia-Lopez and Delgado (2016)

Increased production of wetland rice Boost leaf relative water content, fresh weight and dry weight, decrease in electrolyte leakage, reduced proline content, malondialdehyde and lipid peroxidation, induced heavy metal stress tolerance (Chromium) Increased antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and proline, decreased Na+ accumulation under saline stress and improved mineral content; N (33%), P (48%), K (50%) in saline stressed plants Increased in dehydrogenase and phosphatase Activation of induced systemic resistance (ISR) and plant growth promotion Increased plant growth, increased phosphorus mobilization and uptake (40%), high P adsorption capacity

Hahn et al. (2016)

(continued)

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Table 12.1 (continued) Microbes Bacillus subtilis OTPB1

Bacillus sp. PSB10

Bacillus velezensis LM2303

Bacillus velezensis YC7010

Bacillus amyloliquefaciens p16

Bacillus amyloliquefaciens Bacillus pumilus

Bacillus subtilis BEB-DN

Functional attributes Enhanced systemic resistance and induction of defenserelated enzymes against Alternaria solani and Phytophthora infestans Enhanced growth of plant under chromium stressed soil, ability to tolerate and detoxify chromium metal ion, solubilization of inorganic phosphate Reduced incidence and severity against Fusarium graminearum (Fusarium head blight) Induced systemic resistance against green peach aphid (Myzus persicae) resulted in callose deposition and hypersensitive response Exopolysaccharide produced by bacteria aided in improvement of soil physical structure and enhance water retention capacity Reduced disease severity against Cucumber mosaic virus, Bean common mosaic virus, Pepper mild mottle virus, Tobacco Streak virus, Tomato chlorotic spot virus, Tomato mottle virus, Tomato yellow leaf curl virus Induced systemic resistance against whitefly Bemisia tabaci

Crop plant Lycopersicon esculentum

References Chowdappa et al. (2013)

Cicer arietinum

Wani and Khan (2010)

Triticum aestivum

Chen et al. (2018)

Arabidopsis sp.

Harun-Or-Rashid et al. (2017)

–

Deka et al. (2019)

Cucumber, Cowpea, Tobacco, Cotton, Solanum lycopersicum L. (Tomato)

Jetiyanon and Kloepper (2002), Udaya Shankar et al. (2009), Ahn et al. (2002), Vinodkumar et al. (2018), Abdalla et al. (2017), Murphy et al. (2000), Guo et al. (2019)

Solanum lycopersicum

Valenzuela-Soto et al. (2010)

(continued)

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Table 12.1 (continued) Microbes Ochrobactrum intermedium

Rhodopseudomonas sp.

Rhizobium sp. (strain YAS34)

Bradyrhizobium japonicum

Gluconacetobacter diazotrophicus Pal5

Gluconacetobacter diazotrophicus Pal5

Herbaspirillum seropedicae Herbaspirillum seropedicae DX35

Paenibacillus spp.

Functional attributes Promotion of plant growth and reduced uptake and toxicity of chromium from contaminated soil Enhanced shoot growth, increased fruit/ flower ratio, increase in average fruit weight and lycopene content Solubilization of insoluble phosphate Exopolysaccharides produced by the bacteria modified the soil structure around root system, relieved plant from water stress, increased nitrogen uptake and promoted plant growth Increased in average crop yield and improved nutrient (N, P, Mg) uptake Induced systemic resistance to drought and abiotic stress, increase in productivity parameters, antioxidant defense induction, increase in plant biomass and higher gas exchange and osmoprotectant solutes Increase phosphorus content, higher biomass production, root area and growth index Early lateral root emergence, increased grain production Increase nitrogenase activity and phosphorus solubilization Promoted plant growth, reduced

Crop plant Helianthus annuus

References Faisal and Hasnain (2005)

Lycopersicon esculentum

Lee et al. (2008), Koh and Song (2007)

Helianthus annuus

Alami et al. (2000)

Glycine max

Leggett et al. (2017), Egamberdieva et al. (2018)

Oryza sativa

Filgueiras et al. (2019)

Strawberry (Fragaria spp.)

Delaporte-Quintana et al. (2017)

Zea mays

Canellas et al. (2013)

Oryza sativa

Wang et al. (2014)

Lycopersicon esculentum

Son et al. (2009) (continued)

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Table 12.1 (continued) Microbes

Functional attributes

Crop plant

References

Paenibacillus polymyxa

disease impact and symptom development, antifungal and nematicidal activity Ameliorated soil structure, improved water retention and nutrient transfer Disease control and growth promotion

Triticum aestivum

Bezzate et al. (2000), Gouzou et al. (1993)

Oryza sativa Camellia sinensis

Vidhyasekaran et al. (1997), Saravanakumar et al. (2007) van de Mortel et al. (2012)

Pseudomonas fluorescens Pf1

Pseudomonas fluorescens SS101

Pseudomonas putida

Pseudomonas putida Rs-198

Pseudomonas simiae WCS417r

Pseudomonas fluorescens WCS417r

Pseudomonas fluorescens

Enhanced resistance against Pseudomonas syringae pv tomato and Spodoptera exigua Stable soil aggregate, better soil structure, increase plant biomass, increase survivability under drought stress Increase plant stand and germination, production of phytohormones, i.e., increased IAA and reduced ABA, protection against salt stress Enhanced synthesis of camalexin and aliphatic glucosinolates, induced systemic resistance against leaf chewing herbivore Mamestra brassicae Enhanced attraction of parasitoid wasp Microplitis mediator to Mamestra brassicae (Cabbage moth; generalist insect herbivore) infested plant Elicited priming and induced systemic resistance against herbivorous insects

Arabidopsis thaliana

Helianthus annuus

Sandhya et al. (2009)

Gossypium hirsutum

Yao et al. (2010)

Arabidopsis thaliana

Pangesti et al. (2016)

Arabidopsis thaliana

Pangesti et al. (2015)

Arabidopsis thaliana

Pineda et al. (2012)

(continued)

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Table 12.1 (continued) Microbes Burkholderia megapolitana Burkholderia unamae

Azospirillum amazonense

Paenibacillus polymyxa MAS100

Rhizobium leguminosarum

Functional attributes Antifungal activity, plant growth promotion Expression of ACC (1-aminocyclopropane1-carboxylate) deaminase thereby enhancing plant growth Increased nitrogen accumulation, grain yield and panicle number Suppression of disease symptoms against Ralstonia solanacearum Nitrogen fixation, soil fertility enhancement

Sinorhizobium meliloti

Nitrogen fixation

Bacillus licheniformis Rt4M10, Pseudomonas fluorescens Rt6M10

Acted as stress alleviators against drought by producing abscisic acid (ABA), indole3-acetic acid (IAA)

Algae Spirulina platensis

Spirulina platensis Chlorella vulgaris

Chlorella vulgaris, Spirulina platensis

Enhanced plant growth and seed germination

Promoted growth and yield Increased overall plant growth and decreased soluble carbohydrates Higher amino acid content and mineral composition, increased crop yield

Crop plant Aulacomnium palustre

References Vandamme et al. (2007)

Lycopersicon esculentum

Onofre-Lemus et al. (2009)

Oryza sativa

Rodrigues et al. (2008)

Solanum tuberosum

Soliman (2020)

Lens culinaris Oryza sativa

Hafeez et al. (2000), Kecskés et al. (2016), Rinaudi and Giordano (2010), Russo et al. (2015) Marx et al. (2016), Mitsui et al. (2004)

Alfalfa (Medicago sativa), Medicago truncatula Vitis vinifera

Salomon et al. (2014)

Eruca sativa Ameranthus gangeticus Brassica rapa ssp. chinensis Vigna radiata

Wuang et al. (2016)

Lactuca sativa

Faheed and Fattah (2008)

Vigna radiata

Dineshkumar et al. (2020)

Aung (2011)

(continued)

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Table 12.1 (continued) Microbes Fungi Trichoderma harzianum, Fusarium pallidoroseum

Aspergillus niger, Penicillium notatum

Gigaspora rosea

Penicillium oxalicum

Penicillium chrysogenum

Penicillium sp. EU0013

Penicillium spp. GP15-1

Trichoderma harzianum OTPB3

Functional attributes

Crop plant

References

Increased biomass production, enhanced water stress resistance by induction of antioxidant enzymes Significant increase in soil available phosphorus, increase in phosphorus, nitrogen, protein, oil content and yield of plant Enhanced weight and nitrogen fixation of nodules Promoted plant growth and induced systemic resistance against Sclerospora graminicola (Downy mildew) Increase in seed germination and seedling vigor, activated defenserelated genes against Sclerospora graminicola (Downy mildew) Increased plant biomass, reduced disease severity against Fusarium oxysporum f. sp. lycopersici Increased plant biomass, increased root and shoot growth, reduced disease severity against damping-off Increased root and shoot growth, increased nutrient uptake, enhanced activity of defenserelated enzymes like peroxidase, polyphenol oxidase and superoxide dismutase

Oryza sativa

Gusain et al. (2014)

Arachis hypogaea (Ground nut)

Jitendra et al. (2011)

Glycine max

Sakamoto et al. (2013)

Pennisetum glaucum (Pearl millet)

Murali and Amruthesh (2015)

Pennisetum glaucum

Murali et al. (2013)

Solanum lycopersicum

Sartaj et al. (2011)

Cucumis sativus (Cucumber)

Hossain et al. (2014)

Lycopersicon esculentum

Chowdappa et al. (2013)

(continued)

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Table 12.1 (continued) Microbes Trichoderma harzianum

Trichoderma spp.

Cladosporium sphaerospermum

Alternaria sp. A13

Aspergillus tubingensis, Penicillium sp., Talaromyces funiculosus, Trichoderma harzianum, Trichoderma asperellum Hypovirulent Binucleate Rhizoctonia spp. (HBNR) Trichoderma viride, Chaetomium spp., T. harzianum, T. asperellum Aspergillus niger, Trichoderma koningiopsis

Functional attributes Increased macronutrient uptake, increased seed germination and vigor, induced disease resistance to Plasmopara halstedii (downy mildew) Promoted plant growth and activation of defense mechanism against Xanthomonas euvesicatoria (bacterial spot) and Alternaria solani (early blight) Promoted plant growth, higher amount of Gibberellin production Significantly enhanced dry and fresh plant biomass, increased secondary metabolites accumulation related to medicinal importance Enhanced seed and plant growth along with induced resistance to Colletotrichum capsici causal agent of anthracnose disease

Crop plant Helianthus annuus

References Nagaraju et al. (2012)

Solanum lycopersicum

Fontenelle et al. (2011)

Oryza sativa Glycine max

Hamayun et al. (2009)

Salvia miltiorrhiza

Zhou et al. (2018)

Capsicum annuum

Naziya et al. (2020)

Induced systemic resistance against anthracnose, enhanced lignin deposition and peroxidase activity Induced production of defense-related enzymes, accumulated higher phenolics Activated induced systemic resistance and systemic acquired resistance against wilt disease caused by Rhizoctonia solani

Cucumis sativus

Muslim et al. (2019)

Curcuma longa

Vinayarani et al. (2019)

Triticum aestivum

El-Maraghy et al. (2020)

(continued)

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Table 12.1 (continued) Microbes Acaulospora saccata—A. fragilissima— Scutellospora ovalis—Rhizophagus neocaledonicus— Claroideoglomus etunicatum— Pervetustus simplex Piriformospora indica

Piriformospora indica

Rhizophagus irregularis

Colletotrichum tropicale

Archaea Nitrosocosmicus oleophilus MY3

Mixed species Chara vulgaris— Epiphytic cyanobacteria Claroideoglomus claroideum— Pseudomonas libanensis Anabaena sp.— Trichoderma sp.

Functional attributes Improved fitness and tolerance to metals, reduced translocation of toxic metals to aerial plant part

Crop plant Sorghum vulgare

References Crossay et al. (2020)

Promoted root and shoot growth, expressed droughtrelated genes and conferred resistance against biotic and abiotic stress Promoted plant growth, seed production and increased oil content Enhanced soil nutrient solubilisation and uptake, induced systemic resistance against pathogen Increased lignin and cellulose content leading to reduced pathogen and herbivore damage

Brassica campestris L. ssp. Chinensis

Sun et al. (2010)

Helianthus annuus

Bagde et al. (2011)

Triticum aestivum

Pérez-de-Luque et al. (2017)

Theobroma cacao

Mejía et al. (2014)

Promoted plant growth and induced systemic resistance against necrotrophic bacterium Pectobacterium carotovorum and biotrophic bacterium Pseudomonas syringae

Arabidopsis thaliana

Song et al. (2019)

Nitrogen fixation

Oryza sativa

Ariosa et al. (2004)

Phytoremediation of metal-contaminated soil, enhanced plant growth Increment in available soil nitrogen

Helianthus annuus

Ma et al. (2019)

Chrysanthemum morifolium

Prasanna et al. (2016) (continued)

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Table 12.1 (continued) Microbes

Anabaena sp.— Azotobacter sp. Mesorhizobium ciceri—Trichoderma viride

Aspergillus sp.— Enterobacter sp.

Functional attributes concentration, enhancement in photosynthetic pigments and soil organic carbon concentration Increase in plant fresh and dry weight, higher nitrification potential Accumulation of defense-related enzymes—Lphenylalanine ammonia lyase (PAL), peroxidase (POX) and polyphenol oxidase (PPO). Reduced disease incidence Increased yield and plant growth

Crop plant

References

Chrysanthemum morifolium

Prasanna et al. (2016)

Chickpea (Cicer arietinum)

Das et al. (2017)

Fragaria ananassa

Singhalage et al. (2019)

12.6.1 pH Multiple factors whether biotic or abiotic influence the soil microbial community structure among which the role of pH is significant in the biofilm production. Microbes can tolerate a wide range of pH be it acidic, neutral, or alkaline and therefore, their efficiency in biofilm formation also depends on the optimal pH of the medium they grow on. The microbes with a broad range of pH tolerance soil have a better endurance rate or chance of flourishing compared to microbes having a limited range of pH tolerance. Several microbes belonging to the rhizosphere and rhizoplane of diverse groups of the plant denoted as plant growth-promoting bacteria (PGPB) has been found to be of broader range of pH tolerance (3.0–13.0) and can be utilized for agricultural improvement to enhance plant growth and yield (Kumar et al. 2018). Recent reports showed extreme pH can decrease the growth of certain populations in biofilms (Ansari et al. 2017). Ansari et al. (2017) showed extreme pH negatively affects rhizobia biofilm formation. Similarly, in the case of B. subtilis, growth was not limited in extreme pH and temperature that did not result in pellicle formation. Hostacká et al. (2010) reported that biofilm production of P. aeruginosa increased to 139–244% when subjected to pH 8.5 from 136 to 164% when pH applied was 7.5 in comparison with pH 5.5. Similarly, in the case of K. pneumonia, the production rate increased to 151–319% when pH was 8.5 and production dropped to 113–177% when pH was set to 7.5 compared with pH 5.5. This study also showed that production of V. cholerae non-O1 and O1 biofilm had increased to 204–329% upon pH 8.5 from 123 to 316% while at pH 7.5 as compared with the production upon application of pH 5.5. Hostacká et al. (2010) showed the biofilm

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Fig. 12.1 Chord diagram representing the association between the microbiota and crop plants. The different types of microbes have been clustered and labeled in the figure for easy illustration; the table in the figure represents the abbreviations and their full forms. The common names of the crop plants mentioned in the table has been used to avoid graph layout issue

production increased to about 169% when pH was changed to 7.5 from 5.5, the rise of pH to 8.5 from 5.5 caused an typical difference of 229%. Kumar et al. (2019) revealed differential growth in response to pH were observed in the case of 10 bacterial isolates, which contributed to in vitro plant growth-promoting traits such as phosphate solubilization, nitrogen fixation, production of indole acetic acid (IAA) and siderophore. Some bacterial isolates having plant growth-promoting properties showed positive response under a broad range of pH from acidic to alkaline (about 3.5–12.5) which has the potential use for application as a bacterial consortium in agricultural lands to augment crop productivity.

12.6.2 Temperature and Light Intensity Temperature and light intensity play a major role in several biofilm productions. The fluctuation of temperature and light intensity affect the photosynthetic biofilm production critically. It was reported that a large quantities of specific algal

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assemblies in biofilms were affected due to fluctuations in seasonal light and temperature observed mainly in wastewater treatments (Congestri et al. 2006). Kebede-Westhead and team in 2004 showed that diatoms were more prevalent in low light (270 μmol/m2/s) unlike to high light conditions (390 μmol/m2/s). Similarly, in a work reported by Villanueva and team in, 2011 showed that diatoms contributed to a major biomass fraction under lower temperatures, i.e., 7–11  C in contrast to higher temperatures, i.e., 11–15  C. An increase in temperature from 12, 18, and 24  C increased cyanobacteria proportions in relation to the green algae and diatom populations as cyanobacteria (Schnurr and Allen 2015). This study also showed that application of increased phosphorus and nitrogen loadings, carbon concentrations (inorganic), and also light intensities tend to increase the accumulation of photosynthetic biomass and proportions in comparison to non-photosynthetic biomass.

12.6.3 Oxygen Biofilm formation gets affected under hypoxic conditions in contrast to normoxia (Ghotaslou and Salahi 2013). Among the other key factors, high levels of oxygen activate the production of polysaccharides (Bayer et al. 1990). It has been concluded that polysaccharide concentration follows the similar trend as concentration of dissolved oxygen (DO) within biofilms (Ahimou et al. 2007; Tay et al. 2001). With the increase in the aeration rates, the polysaccharide content has been seen to increase (Tay et al. 2001).

12.6.4 EPS The quality and character of the EPS content of a biofilm may differ depending on the environmental factors (Ahimou et al. 2007). The content of EPS is crucial to cohesion and adhesion of biofilms to the surfaces (Ahimou et al. 2007). The material properties of biofilm such as the strength of the biofilm were largely determined by the EPS (Klapper et al. 2002). Multiple environmental factors also contribute positively to EPS production process. Some of such factors are high availability of oxygen (Bayer et al. 1990), nitrogen limitation (Jarman et al. 1978; Mian et al. 1978), low pH (Ryu et al. 2004), nutrient deficiency (Mao et al. 2001), dehydration (Ophir and Gutnick 1994), and low temperature (Junkins and Doyle 1992). Other than these various other factors such as QS, messenger molecules, salinity and presence of soil enzymes and biocides, also contribute either negatively or positively to the process of biofilm formation.

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271

Biosafety Concern, Regulatory Mechanisms, and Use-Associated Issues

With the increasing food production demand, the rise in the adoption of approaches to improve crop productivity is evident more than ever before. Application of beneficial microbes although still at a commencing stage, its global commercial market is predicted to reach USD 11.6 billion by 2025 (Marketsandmarkets 2020; Singh 2017) because of its deluging potential to complement chemical-based products (Trivedi et al. 2017). This surge for alternative plant growth promotion products has led to a need for risk assessment from the early stage of product development, i.e., isolation of an organism from its natural habitat, to its release and colonization in the targeted regions. Despite its rising popularity, the use of microbes in agriculture has multiple challenges regarding its commercialization, quality control, and standardization of products ((Arjjumend and Koutouki 2018). For example, in the European Union context, there are three strict registration procedures (Regulation (EC) No 1107/ 2009; Commission Regulation (EU) No 283/2013; Commission Regulation (EU) No 284/2013) for plant protection products to reach the commercial market (Lugtenberg 2018). A registration dossier in the EU must submit all the requested information regarding the identification of active microorganisms at its strain level including its biological properties as well as the physical and chemical properties of the product, its safety efficacy, and its safety for humans and the environment. Genotoxicity tests are also to be conducted in vitro and in vivo for mutagenic and carcinogenic studies, respectively. Furthermore, the characterization of the impact on non-targeted organisms especially birds, bees, aquatic organisms, and soil dwellers are observed in laboratory conditions (Pliego et al. 2011). Similarly, in the United States, the federal agencies; EPA (Environmental Protection Agency), and USDA (United States Department of Agriculture) regulate the registration of biostimulants with state-specific mandates and registration compliance (Backer et al. 2018; Du Jardin 2015). Some other countries regard biocontrol agents and biopesticides as pesticides under pesticide acts (Wilson 2003). An example of which is India where the Insecticides Act, 1968, and Insecticides Rules 1971 to a large extent regulate biopesticides and biocontrol agents (Arjjumend and Koutouki 2018). However, the treatment of biofertilizers in India is different. An amendment in 2006 and 2009 on The Fertilizer (Control) Order 1985 has made provisions for addressing biofertilizers (Arjjumend and Koutouki 2017). Apart from this, India also has the guidelines for regulating the export, import, and release of biological control agents and other beneficial organisms approved by the National Plant Protection Organization (NPPO) in 2006. Besides, The Directorate of Plant Protection, Quarantine & Storage on behalf of NPPO is responsible to carry necessary pest risk analysis of the intended import of biological control agent and beneficial organism, before its release and must maintain appropriate records relevant to the organism. Some other country like Canada, for example, groups products based on their intended benefits. Approval of the product is more straightforward if the claimed label on the product relates to

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fertility and nutrient aspects whereas if the product is a biocontrol agent then additional verification and scrutiny will be involved in the process (Backer et al. 2018). Besides these registration and commercialization hurdles, some growthpromoting microbes can never be used as inoculum due to their pathogenic and other detrimental traits over humans, animals, and other plants. Burkholderia cepacia, for example, has several beneficial attributes on plant growth and defense. B. cepacia can induce nodules in crops (Van et al. 2000), has bioremediation properties (Schlömann et al. 1990), and also hold antagonistic properties against several phytopathogens like Meloidogyne incognita (Meyer et al. 2001), Pythium sp., Fusarium sp. (Meyer et al. 2001). Apart from these aforementioned properties, B. cepacia is also involved in infection of cystic fibrosis patients and immunocompromised individuals (Rojas-Rojas et al. 2019; Speert 2001; Tabacchioni et al. 2002). Similarly, several species of bacteria under the genus Stenotrophomonas have plant growth-promoting attributes, but S. maltophilia a plant-associated bacterium can also be responsible for human infection (Coenye et al. 2004; Ryan et al. 2009). For these reasons and to avoid other associated impacts, proper taxonomic position of the microorganism has to be identified before continuing the studies on plant’s performance (Santos et al. 2019). Microbes in general do not exist independently in their natural environmental conditions. But when it comes to evaluating the efficacy of inoculum for plantpromoting attributes, it is based on laboratory screening of pure culture isolate (Ahmad et al. 2008). Only those with better performance are then tested in pot experiments and finally in field conditions (Barriuso et al. 2008). Sometimes those with negligible in vitro performance with alternative promoting mechanisms could be discarded based on classical screening results (Cardinale et al. 2015). Moreover, the effectiveness of microbial inoculation depends on several factors; its colonization efficiency and biocompatibility with the target cultivar, climatic factors, soil properties, root exudates, spatial location, and other living components (Brimecombe et al. 2007; Burns et al. 2015; Rilling et al. 2019), a major aspect of which is studied negligibly (Rilling et al. 2019; Thijs et al. 2016). For developing an inoculum with effective colonization potential in the rhizospheres, it needs to hold a longer self-life and with the appropriate carrier. In fact, for large-scale adoption of microbial products, they need to prove their consistency over a range of climatic and edaphic conditions. “Everything is everywhere, but, the environment selects” (De Wit and Bouvier 2006) a statement by Baas-Becking (1934). The principle behind the old microbiological tenet can set a base to understand how the sole release of a microbe into the environment does not determine the consequences of its release but, the interaction between the released microbe and the environment does (Yoshikura 2015). This consideration is important to study the aftermath of the microbial release. Although the complexity behind the study of such interaction among several ecological components seems exacting, probing plant–microbiome–host interactions seems to be possible by the use of microfluidics-based techniques (Trivedi et al. 2017). This technique can be used to quantify chemotactic processes and responses

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in high spatiotemporal resolutions by the use of a range of optical microscopies ((Massalha et al. 2017; Stanley et al. 2014; Stanley and van der Heijden 2017). Besides this technique, another novel promising method for tracking microbes includes the use of piezoelectric quartz microbalances, where the mass of crystal proportionating to the baseline oscillation can detect target strain (Bunroddith et al. 2018). Such techniques along with omics approach (proteomics, metagenomics, and metatranscriptomics) and next-generation sequencing (NGS) can be useful not only to screen the antagonist strain before its mass production but also to monitor the impact on indigenous microbial communities under field conditions after release ((Lombard et al. 2006; Rilling et al. 2019; Yi and Chun 2015). The rise in the application of biotechnology in agriculture has put light on the use of genetically modified microorganisms (GMM). Although preferentially the use of genetically well-studied microorganisms is chosen for modifications the procedure of GMM use is entangled with several issues, issues that relate to the environment, economics, regulation, and ownership ethics of the product (Nelson 2001). GMM application, no doubt, would raise questions regarding the risk of horizontal gene transfer, the possibility of changing the abiotic environment, and its potential to outcompete/antagonize local microbiota ((van Elsas et al. 2015). The release of a GMM, unlike genetically modified plants, is almost an irreversible process. GMM release is expected to have a functional and structural shift where the indigenous microbes’ community has low resiliency, but in a robust community, the release of GMM would have a negligible impact on the dominant microbial community or microbiological soil functions (Tebbe and Miethling-Graff 2006; Tebbe 2015). Therefore, while carrying environmental risk assessment considerations on the indigenous microbial community’s resiliency is crucial.

12.8

Keyword Mining

With the exponential increase in the literature data, the requirement to extract meaningful information automatically has been indispensable as handling these massive data can be really cumbersome. Though the data analytic approaches are in the developing stage, there are still a good number of tools and methods to mine text data to understand the basic nature such as topic analysis, keyword frequencies, and much other similar information. The mining of published articles to evaluate the research productivity using different bibliometric indicators as well as understanding the structure with the help of topology of tokens (which are basically pre-processed important words in the articles) has obtained an upsurge interest in the last decade. The keyword-based text-mining frameworks are very commonly used ones with simple analysis methods which can be easily applied to most type of articles and also proven to be of great help to develop systematic reviews (Li et al. 2016). To have an idea about the publication patterns and research outputs, we performed a basic scientometrics analysis. A search on PubMed with the query term < “biofilm” AND “agriculture” > retrieved a total of 1018 records. In order to get a clearer view of the subject of the PubMed reports, a simple Venn diagram was

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Fig. 12.2 Venn diagram based on keyword mining

created with the help of Venny (Oliveros 2015) based on the output of this scientometrics analysis (Fig. 12.2). The Venn diagram is based on the mention of the biofilm-producing microbial taxa that have been found in the PubMed reports based on the simple keyword searches. It can be seen that out of the 1018 reports, the highest mentioned category was bacteria followed by fungi, algae, and archaea. The idea was to obtain keywords by mining the titles and abstracts of the related articles on PubMed and infer into the specific areas in agriculture where biofilms are being used. The PubMed identifiers (PMID) were downloaded and parsed through an R script to create a word cloud for the frequent keywords. The PMIDs were used as input and with the help of the RISmed package, the titles and abstracts were retrieved (Kovalchik 2017). The widely used text-mining package tm was used to create a corpus out of the titles and abstracts of all the records (Feinerer et al. 2008). The corpus was pre-processed to get rid of noises such as stop-words, punctuations, and extra spaces. A term “document matrix” was created using the same package following a record of the words and its corresponding frequencies. Finally, a word “cloud” was created keeping the frequency of the words as a metric reflected with the font size directly proportional to the frequencies (Fig. 12.3). The word “cloud”was restricted to the most frequent 500 words to get a clear picture of the over-represented words in the articles. There are still some unnecessary words which are trivial English words which can be ignored while trying to note the

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Fig. 12.3 Word Cloud based on keyword mining

research areas. Although some of the words such as “colonization,” “surface,” and “extracellular” are frequency wise less in number but can be seen due to the relevance to the subject of the study conducted. As expected, the most frequent word was “biofilm” with a total frequency of 2774. A redundant word which is “biofilms” again came in the third position with 1044 occurring. The second frequent word was “formation” which must be due to its frequent association with the word “biofilm.” The places from fourth to eighth contained words “bacterial,” “strains,” “study,” “bacteria,” “strain,” respectively, suggesting that the bacterial biofilms are the most frequent ones that have been used in the field of agriculture. This purely coincides with the fact that the bacterial biofilm reports are abundant as mentioned above in the Venn diagram.

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Conclusion

The current scenario with respect to concurrent events of the gradual loss of agricultural lands and increasing food demand to fulfill the need for the population outburst is paving the way to look for sustainable alternatives. Biofilms produced by agriculturally important microbes hold a very important space in promoting sustainable agricultural practices. The current research scenes in this old yet budding concept in terms of applicability shows much more focus on bacterial biofilms whereas other biofilm-producing microbial taxa should also get equal attention. It surely will help in the extraction of novel information and thus strengthen the knowledge base. There is a need to increase the funding by the government in this sector and more efforts to popularize the applicability of the beneficial biofilmproducing microbes to uplift the crop and soil health keeping the probable harmful effects in check.

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Biological Soil Crusts to Keep Soil Alive, Rehabilitate Degraded Soil, and Develop Soil Habitats

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Atoosa Gholamhosseinian, Adel Sepehr, Behnam Asgari Lajayer, Nasser Delangiz, and Tess Astatkie

Abstract

Soil is a mixture of liquids, gases, minerals, organic matters, and microorganisms that support life together and play a vital role in the ecosystem cycles. Soil is an important part of agriculture, which is a major source of food. Healthy soils produce plenty of healthy nutrients. Adding chemical fertilizers has been considered as a good solution to increase agricultural production and secure human needs for food for many years. As a result, excessive use of these fertilizers has led to soil contamination and reduced fertility. The use of chemicals has also endangered the environment, and to mitigate this, using environment friendly biofertilizers have been considered. Soil biocrusts, which include cyanobacteria, lichens, mosses, and fungi, live on the surface and inside the soil. Biocrusts play an important role in ecological remediation to increase soil water, dust capture, interaction with vascular plants, increase soil stability, reduce water and wind erosion, fixing N and P, etc. In this chapter, what biological crusts are and their

A. Gholamhosseinian · A. Sepehr Department of Desert and Arid Zones Management, Ferdowsi University of Mashhad, Mashhad, Iran B. Asgari Lajayer (*) Health and Environment Research Center, Tabriz University of Medical Sciences, Tabriz, Iran e-mail: [email protected] N. Delangiz Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran T. Astatkie (*) Faculty of Agriculture, Dalhousie University, Truro, NS, Canada e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_13

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role in improving soil conditions, reducing erosion, and their use in plant germination, and as biofertilizer is reviewed. Keywords

Aggregate stability · Biocrust · Biofertilizer · Seed germination

13.1

Introduction

The current population of the world is 7.6 billion, and at the current rate of population growth, the population of the world is forecasted to reach 9.8 billion in 2050, and 11.2 billion in 2100. To meet the needs of this growing population, the production of agricultural products including cereals needs to be increased, but this is becoming unattainable due to land and soil constraints, which means that there is a strong need for solutions to feed the growing population of the world. This goal of achieving food security is primarily the responsibility of the agricultural sector. This target can only be achieved by converting more land for cultivation or increasing the productivity of available cultivated land. Since the first option is not feasible due to land constraints, increasing the productivity of agricultural products is the only viable option. The best ways of increasing agricultural productivity are improving soil fertility and using environment friendly tools. At present, agricultural production is dependent on chemical fertilizers, pesticides, over-irrigation, etc. These practices have caused environmental and health problems including loss of soil fertility, overuse of water and land resources, reduced plant diversity, and environmental pollution (Singh and Strong 2016). Sustainable agricultural practices can solve all problems related to food production and issues related to quality and environmental health (Mason 2003). Among the sustainable agricultural practices, the use of biofertilizers is one of the best. Biofertilizers are living microorganisms that can secrete certain organic compounds into the soil that increase seed growth, plant growth, and soil fertility. Early living entities on an evolutionary path of indefinite duration led to the creation of the last common ancestor that had the basic characteristics of all cells. Early life evolution was accompanied by metabolic diversification, and as soon as life appeared, it started to evolve and diversify (López-García and Moreira 2006). Biological soil crusts (BSCs/biocrust) are the pioneers of life on Earth and include cyanobacteria, green algae, moss, and lichens (Belnap et al. 2006), and they occupy the surface and the soil in the top few millimeters (Warren et al. 2019). In recent years, considering the importance of replacing chemical fertilizers by biofertilizers for the creation and development of sustainable agriculture, studies on the effects of biocrust have increased. Furthermore, biocrust microbes are the most dominant community types (Zaady et al. 2013), and one of the first forms of earthly organisms whose life probably dates back to 2.6 billion years. Biocrust microbes can be found in different ecological, succession, and agronomic conditions where infertility resulted in colonization opportunities of bare soil surfaces (Elumeeva et al. 2011). Biocrusts are more common in arid and semi-arid ecosystems, where diversity and vegetation cover are low.

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Communities dominated by mosses, lichens, green algae, and cyanobacteria are biological keys to polar, temperate, and arid ecosystems worldwide, where they have key roles in controlling ecosystem structure and function (Belnap and Lange 2003). However, there is very limited information on the potential effects of different species on soil performance and microbial communities.

13.2

Cyanobacteria and Green Algae

Algae are found all over the world in a variety of environments such as freshwater, seawater, snow, rock, and on the surface of plants and animals. Many algae, such as Oscillatoria Brevis, Scytonema elongates, Heterhormogonium, can tolerate temperatures between 50 and70  C, which is making them common in tropical soils (Fig. 13.1). Although many algae can tolerate a lot of sunlight, their growth rate is low under light conditions. Many green algae can be found in a wide range of pH (Heidarpour et al. 2019); however, blue-green algae prefer neutral or alkaline pH (Bilgrami and Saha 2004). After the death and decomposition of algae, organic matter is added to the soil, and after some years, the soil becomes suitable for cultivation. Cyanobacteria species that are found all over the world in fresh and saline waters, in temperate, arid, and tropical regions, on tree trunks, and in soils as microbial communities (Thajuddin and Subramanian 2005) play a vital role in soils’ physicochemical properties. They are also able to occur in all terrestrial and aquatic habitats,

Fig. 13.1 Cyanobacteria and algae cultivated in the semi-arid region of northeastern Iran. (a) Osillatoria sp., (b) Phormidium sp., (c) cyanobacteria and green algae, (d) Coccomyxa sp., (e) Phormidium sp

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such as oceans, rivers, freshwater, wet rocks, and bare soil, or in contact with other plants and animals such as lichens, sponges, and Protestants (Vaughan 2011). Cyanobacteria are photosynthetic and can manufacture their own food. They are more than 3.5 billion years old fossils (Soudzilovskaia et al. 2011). It is understood that cyanobacteria played an important role in producing oxygen for the atmosphere and oceans (Demoulin et al. 2019). Cyanobacteria are prokaryotic phototrophs that have been considered in many industries, including food supplementation and the production of some valuable essential polymers (Arias et al. 2020). It is also used for various purposes, including dietary supplements and food, due to its high content of vitamins and proteins, as well as its high digestibility (Abed et al. 2009; Rosgaard et al. 2012; Colica et al. 2014). Cyanobacteria have played an important role in early evolution and life on Earth. Moreover, cyanobacteria can grow and survive in unfavorable conditions for a long period of time when environmental conditions are not conducive to growth (Whitton 2000). The high compatibility of cyanobacteria with any environment indicates that their culture, like other microorganisms, does not require energy-rich compounds (Lau et al. 2015).

13.3

Mosses

Mosses are groups of the simplest early plants. These plants have an intermediate position between green thallophytes (algae) and vascular plants (Jackson et al. 2011). This slow-growing plant is one of the first terrestrial plants and does not have stem organs, leaves, and roots, but has similar structures. Plant stabilization, soil uptake, aggregate retention, and uptake by a filamentous structure called the rhizoid, which consists of several brownish-red cells (Fig. 13.2c). The impact of mosses on the inorganic environment is substantial (Cornelissen et al. 2007) as they control the temperature and moisture fluctuations in soils (Bueno et al. 2016; Gornall et al. 2007; Jackson et al. 2011; Soudzilovskaia et al. 2011). Mosses can also affect the availability of soil nutrients (De Long et al. 2016) by affecting soil temperature, reducing albedo, and improving substrate quality (Gornall et al. 2007; Lang et al. 2009). Moss species have many effects on soil properties that can improve the environment for the growth and establishment of vascular plants (During and van Tooren 1990; Sohlberg and Bliss 1987). Studies have shown that moss species have different establishment and seedling responses to warming or rainfall when other factors are kept constant (Lett et al. 2017; Stuiver et al. 2014).

13.4

Lichens

Lichens are a section of the plant flora in many parts of the world; they were the first multicellular creatures to settle on land (Clauzade and Ozenda 1970). Lichens are complex and highly developed plants. Lichens have high endurance due to photosynthesis and stabilization of solar energy by algal part and resistance of fungal

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Fig. 13.2 (a) Lichen (Xanthoria parietina) with mosses; (b) Syntrichia sp.; (c) The morphology of moss consisting two parts: sporophyte (1,2) and gametophyte (3), 1. Capsule 2. Seta 3. leaf 4. Rhizoid; (d) Moss grown on rock in the semi-arid region of northeastern Iran

filaments in harsh environmental conditions and can survive at minus 60  C. Lichens are living organisms composed of mainly mycobionts and photosynthetic algae and/or cyanobacteria (photobionts; one species of mycobiont (fungi) and one species of photobiont (algae)) form a symbiotic relationship in a thallus. The body of lichens is composed of several layers: upper hard layer of fungal filaments, algae layer that is mostly made of algal or cyanobacterial cells, called the medulla, the central part, which consists of fungi filaments and some of the materials made by lichen, is stored in this part, and the lowest and thickest layer is made of fungal threads with a thickness of 0.5 mm. Soil living lichen subspecies (e.g., Lepraria crassissima and Diploschistes diacapsis) have higher respiration rate, enzyme activity, and concentration ability of the inorganic soil in comparison with cyanobacteria living soils (Kroken and Taylor 2000; Muggia et al. 2010; Miralles et al. 2012, 2013) (Fig. 13.3).

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Fig. 13.3 (a, b) View of lichens in the northeast of Khorasan Razavi province; (c) view of terrestrial lichens (psora sp.); (d) the red circles show the soil lichens and the blue circles show the mosses; (e) xanthoria parietina; and (f) an example of lichens on a tree trunk

13.5

Ecological Roles of Biocrusts

Studies have shown that biocrusts play a significant role in ecological remediation. There are many ecological roles of biocrusts: (1) the redistribution of precipitated rainwater; (2) the capture, collection, and use of airborne and soil nutrients; (3) interaction with vascular plants; and (4) enhancing the soil physical structure and stability; (5) reducing the wind and water erosion; (6) increasing nutrients accessibility; (7) increasing the roughness of the surface, so enhancing dust trapping capacity (Fearnehough et al. 1998; Belnap and Lange 2003; Warren et al. 2019; Weber et al. 2016); (8) improving the essential nutrient concentration in plants (Belnap and Harper 1995); (9) enhancing nitrogen and carbon fixation rate (Evans and Lange 2001; Yang et al. 2014); so, increasing the mineralization and bioavailability rate of nitrogen (Delgado-Baquerizo et al. 2013; Hu et al. 2015); thus, biocrusts nutrient enrichment has an important role in soil fertility and nutrient bioavailability. Also, it is important in the biogeochemical cycles.

13.5.1 The Role of Soil Microorganisms in Inhibiting Runoff Runoff is one of the important research topics in hydrology (Martinez-Hernández et al. 2017). Erosion determines the rate of sedimentation as well as plant establishment (Le Bissonnais 2016), and it is considered as a valuable tool for studying the structure, distribution of plants, and the function of ecosystems (Neilson 1995).

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Microorganisms such as earthworms, insects, bacteria, mosses, cyanobacteria, algae, fungi, and lichens are accumulated on the surface of soils with several millimeters of thickness. As ecosystem engineers, they form the biological surface of the soil thus providing the ground for the growth and development of their space by changing soil surface conditions that has an impact on other animal, plant, and soil habitats, as well as combating soil erosion. One of the most important functions of the biological crust is soil stabilization, which leads to a reduction in water and wind erosion (Williams et al. 1993). Soil microorganisms are useful and efficient agents to improve the quality and fertility of soils (Saghafi et al. 2019a, b, 2020). They also decrease soil degradation (Bowker 2007; Rossi et al. 2017; Maestre et al. 2017). It was suggested that inoculating biological crusts that manipulate soil communities could be a key action to restore damaged ecosystems (Wubs et al. 2016). A study on the Colorado Plateau showed that under rainfall simulation conditions, in samples inoculated with Cyanobacterium, sediment erosion of 400 g/m2 and samples containing lichens and mosses were almost zero (Belnap et al. 2012). Also, a 100% decrease in water erosion was observed in the Loess Plateau Region, in the presence of moss crust relative to bare soil (Zhao and Xu 2013).

13.5.2 Hydrology and Available Soil Water Soil moisture plays a key role in the rate of residue decomposition and mineralization of organic phosphorus. There are obvious contradictions about the role of biocrusts in relation to their effects on soil hydrology (effect on runoff infiltration). Several researchers concluded that biocrusts increases penetration, while some believe that the presence of the biocrust reduces it. Climate change influences the way in which biocrust affects hydrology, and how it increases runoff (and not sediment loading process). It may also increase infiltration (Chamizo et al. 2016). An extensive review by Warren et al. (2019) revealed that much of the variation can be attributed to soil texture. When the content of sand in the soil exceeds about 80%, and the soil is not frost-heaved, biocrusts generally reduce water infiltration compared to soil without crust. This seems to be attributable to soil porosity. Porosity in sandy soils is possible with the accumulation of biocrust in finer soil particles when polysaccharides exudates, root structures, fungal hyphae, or cyanobacterial filaments block the pores of the soil, preventing water from moving and penetrating (Rodriguez-Caballero et al. 2018). When sand content is less than about 80%, biocrust organisms contribute to soil aggregation, and, as a result, it leads to higher rates of porosity and more penetration than soil without biocrust. Biocrusts increase the surface roughness, which increases the retention time of water on the surface (Faist et al. 2020), it also affects the movement and retention of water in the soil. The process of runoff production and re-infiltration plays an important role in ecosystem efficiency and large-scale hydrological response (Kidron 2016). When the biological crusts get wet, their volume increases up to ten times, which results in the absorption and retention of water in the soil.

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13.5.3 Application of EPS on Improving Soil Properties Exopolysaccharides (EPS) are the most vital chemicals that are synthesized by cyanobacteria. They play an important and vital role in improving soil parameters (Weber et al. 2016). These biopolymers form a layer around cells called capsule (De Philippis and Vincenzini 1998). It protects cells that are under physical stress such as drought (Costa et al. 2018). Exopolysaccharides regulate the function of cell loss or water absorption (Mazor et al. 1996; Potts 1994; Adessi et al. 2018) to protect cells from harmful effects of dehydration-hydration cycles and environmental stresses (Liu et al. 2016). One of the most important studied roles of the EPS matrix is the accumulation capacity of soil particles that is important for improving soil structure and fertility (Boonchai et al. 2014). EPS acts like a glue bond to clay and ions and holds soil particles together (Chenu 1995). These exo-polymers that are studied thoroughly can improve the quality, fertility, and structure of soils (Hwang et al. 2004; Elisashvili et al. 2009). EPSs are produced in response to environmental stresses such as salinity, pH changes, drought, temperature changes, etc., to protect the cell (Wingender et al. 1999). EPSs also enhance water retention and maintain moisture for a longer time (Rossi et al. 2012; Colica et al. 2014; Adessi et al. 2018) and defend microorganisms from dehydration and nutrient constraint to help them to survive (Zhang 2005; Parikh and Madamwar 2006). EPS produces a layer around microbes that protects them from the negative effects of heavy metals and antimicrobial chemicals. EPS matrix can also protect cyanobacteria and the environment against drought by retaining water. EPSs are widely used in the food and agricultural industries. Their glycosidic linkage is a kind of gelling or thickening agent (Kraan 2012). Also, EPS can play the role of water binding, stabilizer, or emulsifier agent in food industries (Whistler and Daniels 1990; Singha and Hemachandran 2012). Moreover, EPSs are kind of antibacterial agents, and anticancer (polysaccharide from Spirulina sp., inhibits tumor incursion), antiinflammatory, and immunomodulatory agents (Kraan 2012; Orsavova et al. 2015; De Jesus Raposo et al. 2015). Also, EPS secreted by Cyanobacterium aponinum sp. (Gudmundsdottir et al. 2015) and Lyngbya sp. are known to serve as an immune system booster and used in pharmaceuticals (Pignolet et al. 2013; Liu et al. 2016; Raposo and Stoorvogel 2013). Chlorella sp. and Spirulina sp. are rich in vitamins and proteins, so they are important in the nutraceutical industry (Choi et al. 2013).

13.5.4 Biocrust and Soil Nutrition The activity of microorganisms in poor soils depends on conditions such as temperature, humidity, and the presence of organic carbon compounds. During the process of photosynthesis, the autotrophic activity of biocrust causes carbon dioxide to be absorbed by the air and stabilized in the soil as carbohydrates and polysaccharides. EPS is essential for microbial life because it traps nutrients, provides a suitable environment for chemical reaction, and protects microbes from stresses such as

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drought and saline soils. As microbial EPS improves the aggregation ability of soil particles, it can increase the water retaining and nutrients trapping capacity. Whereas Cyanobacteria have the ability of N2 and CO2 stabilization, these biocrusts can stabilize a wide range of compounds such as P and vitamins (Priya et al. 2017). Thus, they can improve the fertility of soils in arid and semi-arid regions, where water and N are the most limiting factors (Noy-Meir 1973). Studies in the Sonoran Desert lowland and the Colorado Plateau highland indicated coincident trends of biogenic elements –N and C- enrichment and the non-biogenic elements, such as Zr, AS, Zn, Cu, Ca, Mn, and Cr depletion (BeraldiCampesi et al. 2009). The biocrusts physiological and survival activities depend on many elements. Also, the composition of macro- (Ca, Mg, N, P, and K) and microelements (Fe, Zn, Cu, and Mn) is related to the composition and distribution of biocrusts in soil (Bowker et al. 2016). For instance, D. diacapsis can increase the concentration of micro- and macro-elements in comparison with the bare areas without lichens (Concostrina-Zubiri et al. 2013). Other studies showed that lichens and mosses have positive correlations with manganese, zinc, potassium, and magnesium, but negative correlation with phosphorus (Bowker et al. 2005; Ochoa-Hueso et al. 2011). Housman et al. (2006, 2007) and Li et al. (2010) reported that microbial biomass and nutrient concentration of the moss-covered regions is higher than regions with cyanobacterial crust. Soils with higher carbonate content are suitable for the growth and establishment of lichens and mosses. All these functions help to improve the dust capture capacity and the accumulation of essential elements and organic matters. Thus, the development of the soil surface and biocrusts formation will occur (Li et al. 2006).

13.5.5 Soil Texture and Aggregate Stability Soil texture is the coarseness or fineness of the particles, which is mainly obtained from the relative proportions of different particles such as silt, sand, and clay. Soils are the product and result of the performance of various processes, which are formed over time genetic horizons. The location of soil formation, intensity, and weakness of influential factors such as time, determine the speed of soil formation and evolution. Other factors that affect soil formation are bedrock material, weathering processes, mineral composition, and type and composition of organic matter. Soil texture has a great impact on soil physical and chemical performance (Bowman and Hutka 2002) such as water and nutrient mobility, air availability, and soil water content. Cyanobacterial crusts with the same species are concentrated in fine-textured soils closer to the surface than in coarse soils because light and moisture are rapidly limited in depth (Garcia-Pichel and Belnap 1996). Polysaccharides can bond with soluble minerals when soils with a weak structure are wet and after drying. In finetextured soils, the recovery rate of cyanobacteria is faster because they require less energy for colonization and accumulation due to smaller soil pores (Zheng et al. 2011; Rosenstein et al. 2014); as a result, they are easily attached to soil aggregates

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a

b

100µm EHT = 20.00 kV WD = 7 mm

Signal A = QBSD Photo No. = 4033

Date :27 Aug 2018 Time :18:21:15

20µm EHT = 20.00 kV WD = 7 mm

Signal A = QBSD Photo No. = 4028

Date :27 Aug 2018 Time :18:08:40

Fig. 13.4 Electron microscope images are taken from the cross-section of biocrust soils, showing hyphae and polysaccharide filaments of mosses and lichens (red arrow) that have kept the soil cohesive (LEO 1450VP, Germany-central lab in Ferdowsi University of Mashhad)

through the secretion of exopolysaccharides (Chock et al. 2019). The decaying material mixes with the soil and acts as a glue to bind the particles. Increasing humus in the soil is important to maintain moisture. Algae and cyanobacteria synthesize extracellular polymeric materials (EPS), which contain various biopolymers and help EPS to bind to the surface of minerals (Flemming and Wingender 2010). In most soils, especially in arid and desert areas, cyanobacteria are the predominant species in the biological crust and provide an environment where other biological crust components can be located (Abed et al. 2009). So, the filaments of the cyanobacteria can facilitate the formation of a stable soil surface through binding to the soil particles. These stable surfaces make a suitable environment for organisms such as lichens and mosses to colonize and develop (Deng et al. 2020). The Scanning electron micrographs (SEM) shown in Fig. 13.4 has details of the internal structure of soil crusts and the role that cyanobacteria play in soils as well as the infiltration of fungal hyphae in the soils. According to research conducted by Wang et al. (2017), in the Atacama Desert, biocrusts are able to protect soil from wind erosion as well as to increase soil cohesion by trapping dust and atmospheric salts. Sepehr et al. (2018) examined the relationship between cyanobacteria and soil parameters and found that cyanobacteria increase organic carbon and nitrogen and their EPS secretions cause more stability in aggregates. The effect of biocrusts on the amount of water in a region of southwestern Spain was studied using a pressure plate device at two pressures of 33 and 1500 kPa and in two layers of 0–1 and 1–5 cm covered by crusts. They observed a significant increase in the amount of water at a pressure of 33 kPa in the order of the least developed crust, in the direction of the crust with the most developed in all layers, and at a pressure of 1500 kPa the types of crusts had a similar amount of water (Chamizo et al. 2018).

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Sustainable Agriculture

13.6.1 Wastewater Treatment Domestic, agricultural, and industrial wastewaters contain high concentrations of phosphorus and nitrogen, which may lead to eutrophication and toxicity (Pouliot et al. 1989; Magro et al. 2012). Over the past few decades, efforts have been made to use microalgae for secondary effluents’ biological treatment (Oswald and Gotaas 1957; de la Noüe et al. 1992; Heidarpour et al. 2019). Cyanobacteria were successfully used in the treatment of municipal, livestock, and industrial wastewater and has met with considerable success (Muñoz and Guieysse 2006; Lee et al. 2015). Cyanobacteria can be widely used in wastewater treatment by removing nutrients, reducing biological oxygen demand (BOD), and chemical oxygen demand (COD), as well as having a high ability to remove heavy metals (Abdel-Raouf et al. 2012; Honda et al. 2012). Cyanobacteria are one of the largest groups of Gram-negative prokaryotes that have great diversity in morphology, cell division patterns, physiology, cell differentiation, and habitats (Rippka et al. 1979). In general, cyanobacteria have different properties that make them a good option for wastewater treatment. The capacity and ability of cyanobacteria to grow in various effluents such as municipal and industrial wastewater has already been studied (Parmar et al. 2011; Vijayakumar 2012). The use of cyanobacteria in wastewater treatment, in addition to economically valuable by-products, has also been considered to produce clean- or at least cleanerwater (Markou et al. 2014). Cyanobacteria can accumulate phosphorus and mineral nitrogen store it in the form of polyphosphate and cyanophycean and use it for growth (Fay 1983; Abdel-Raouf et al. 2012). Cyanobacteria can survive and thrive in highly variable conditions with contaminated effluents. Another advantage of filamentous cyanobacteria (epilithic or benthic) is their natural tendency to aggregate and precipitate easily in the absence of stirring, which makes them excellent candidates for mass cultivation for wastewater treatment (de la Noüe and Proulx 1988; Mespoulède 1997). During numerous studies, the ability of cyanobacteria to biodegrade and bio-sorb persistent heavy metals have been demonstrated (Kirkwood et al. 2001; El-Bestawy 2008). Their minimal nutrient needs have made them suitable for using them as low-cost, environment friendly technologies for wastewater treatment as well as to produce biological products (Lau et al. 2015). Blue-green algae are known to store polymeric compounds such as polyhydroxyalkanoates (PHA) and carbohydrates under environmental stress and nutrient deficiency conditions (Samantaray et al. 2011). PHA has received a lot of attention due to its potential use as a substrate for bioplastics and biofuels. This type of wastewater treatment is an economical and environment friendly process without pollution (Honda et al. 2012; Rawat et al. 2011).

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13.6.2 Seed Germination and Establishment of Vegetation Biocrust can lead to stunted or optimal plant growth (Condon and Pyke 2018a; Havrilla et al. 2019); the biocrusts may act as armor coatings to prevent seed penetration, but if the seeds can be successfully established, the presence of biocrusts can increase seedling growth rate (Ferrenberg et al. 2018; Slate et al. 2019). The positive effect of cyanobacteria on the germination and growth of wheat and rice seeds has been reported (Muñoz-Rojas et al. 2018). Furthermore, the use of beneficial microorganisms such as cyanobacteria on the seeds can be a useful tool for transferring inoculation to the soil after germination (O’Callaghan and van Sinderen 2016). Soaking the seeds in cyanobacterial suspension allows them to be impregnated into the seed to create desirable conditions for inoculation (Mahmood et al. 2016). Research has shown that in paddy fields, cyanobacteria help to accelerate seed germination and to promote seedling growth (Gupta and Lata 1964). This research showed that cyanobacteria are richer in protein and therefore have a positive effect on grain yield and quality. The potential of cyanobacteria as a biological primer to restore soil in an arid region of Western Australia was investigated, and the results showed positive effects on the growth of native plants and on the improvement of soil substrate characteristics (Flemming and Wingender 2010). Cyanobacteria have more than 800 secondary metabolites including linear, cyclic peptides, fatty acid, ribosomal peptides, and amides (Costa et al. 2008). In addition, they produce various compounds in soils that can improve soil fertility. They are also known as biocontrol against fungi and harmful bacteria for plants. Some of these include polypeptides, proteins, vitamins, biotins, amino acids, and growthpromoting factors (Singh et al. 2016). This secondary metabolite shows different clinical activities such as antimicrobial, anticancer, antitumor, and anti-inflammatory (Nagarajan et al. 2012). Cyanobacteria are able to exude plant growth regulators (PGRs) (Venkataraman 1981); Cytokinin-like (Strick et al. 1997) and gibberellins-like compounds (ShenRui and Shen 1997) are some of them. Furthermore, cyanobacteria exude pharmaceutically active compounds (Metting and Pyne 1986; Chamizo et al. 2020), and organic acids, toxins, antibiotics, and algicides (Hellebust 1974). The Chemicals produced by cyanobacteria have essential roles in regulating metabolism, plant growth, development, and improving germination (Hashtroudi et al. 2013). Cyanobacteria can lead to favorable microhabitats for soil and other plants, and also improve the establishment and growth of annual plants (Xu et al. 2013; Samantaray et al. 2011). It was also reported that the use of cyanobacterial extract increased the growth of plants as well as their dry weight (Offer et al. 1992). The use of dilute aqueous extracts of cyanobacteria was successful in the control of a damping-off disease caused by fungal strain (Caire et al. 1976). The use of cyanobacteria to increase seed germination is recommended as a biological fertilizer and to increase the growth of many plants (Strick et al. 1997). After regeneration and establishment of vegetation, experimental findings showed that the concentrations of essential elements in the soil with

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cyanobacterial-lichen crust, moss crust increased significantly. Therefore, plant revegetation also has a mutual effect on the development of cyanobacteria-lichen and moss crusts. Due to these characteristics, their high dispersion and establishment in any environment, cyanobacteria can be known as ecosystem engineers and can be used as a potential tool for soil repair in arid areas (Rossi et al. 2017).

13.6.3 Biofertilizer Biofertilizers are good replacements of chemical fertilizers due to their environmental compatibility, because of which they have received a lot of attention in recent decades. The widespread use of chemical fertilizers and pesticides for a long time led to the contamination of soil (Khoshru et al. 2020; Asgari Lajayer et al. 2019). Recently, agriculture has relied on chemical fertilizers to increase yield, which also reduces soil microbiota and thus reduces soil fertility (Singh et al. 2016). It has been found that biological soil crusts contribute to soil fertility resulting in increased crop yield and reduced pollution of the environment, water, and soil (Rossi et al. 2017). Soil fungi are one of the main pathogens in crop fields. Kulik (1995) stated that cyanobacteria have a high yield as a biological fertilizer for plant pathogenic bacteria and fungi. Anabaena and Nostoc species are two of them that are considered as biofertilizers (Li et al. 2002). Nostoc sp., Anabaena sp., and Oscillatoria sp. have a high abundance, especially in arid areas and can produce a lot of EPS and survive during environmental stresses.

13.6.4 Biocrusts Functions and Utility in Restoration Arid and semi-arid lands are the most extensive biomes on Earth, covering about 45% of the surface of Earth (Reynolds et al. 2007; Pravalie 2016). Biocrusts are able to live in areas that are difficult for vascular plants to survive, and they work on basic functions of ecosystems such as protecting soil from erosion, conserving moisture adjustment hydrology, and providing nutrient cycling of carbon and nitrogen fixation (Belnap et al. 2016). Restoration of biocrusts in arid and degraded areas should be a top priority because the regeneration of biocrusts leads to the recovering of many desired environmental functions (Bowker 2007). During the biological crust development process, the transfer of dominant functional groups from cyanobacteria to mosses and lichens changes the structure of bacterial communities because of the different effects of nursing on soil bacteria (De Velasco et al. 2017). However, knowledge about the role and diversity of the bacterial community in successive biological sequences in environmental regeneration remains limited (Condon and Pyke 2018b; Liu et al. 2016). Recently, studies showed the role of biocrust functions in the ecological restoration of the ecosystem of deserts (Bowker 2007; Winkler et al. 2018), and the critical role they play in predicting desert ecosystem responses in different climatic conditions (Weber et al. 2016). Cyanobacteria EPS properties are important factors for selecting the type of

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crust for soil restoration. Specific properties of cyanobacterial EPS in terms of monosaccharide and macromolecular composition can provide useful understandings in this area.

13.7

Conclusion

Biological soil crusts have an important role in physical and chemical properties of soil, particularly in arid environments, although most of these effects are magnified on the surface layers (0–5 cm). Biological soil crusts are ecological engineers in nature and their positive activities in the soil introduced them as an indicator of soil quality. Biological soil crusts conserve soil and protect it against erosion. This important role led researchers to utilize them for erosion control and actions related to soil restoration and combating desertification. They have an essential role in soil stability, regulation of salinity, water balance, and fertility enhancement particularly in arid ecosystems and environments with hyper conditions regarding climate and erosion.

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Fungal Chitosan: The Importance and Beneficiation of this Biopolymer in Industrial and Agricultural Process

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Allwin Ebinesar, Veena S. More, D. L. Ramya, G. R. Amrutha, and Sunil S. More

Abstract

Chitosan is used as an alternative effective bio-pesticide to manage the crop diseases and to increase the fertility of soil surface. Chitosan is a chitin derivative composed of poly(β-(1–4)-D-glucosamine) isolated from fungal cell walls, crustacean, and shrimp shell. It is a polymer that is non-toxic, biocompatible, and conveniently biodegradable. Chitosan is capable of persuading callose formation in plants, by synthesising phytoalexins to inhibit the growth of parasites in plant cells. In addition, it enhances stomatal conductance and decreases the phase of transpiration without affecting the plants’ physical nature. Furthermore, to alter the permeability of the membrane, Chitosan is used as a coating agent for nuts, fruits, and vegetables. It raises sugar and proline concentrations and increases the activity of peroxidase, catalase, phenyl aniline ammonia lyase, and tyrosineammonia lyase enzymes. It also has the potential to release the fertiliser in a continuous manner, and it increases the soil water retention property. In addition, chitosan has a high potential to resist pathogenic fungus formation, sporulation, viability of spores, and fungal germination. For four decades, chitosan has been used in a variety of applications for various applications, such as enzyme immobilisation, enhancement of healing activity, heavy metal removal, industrial residue treatment, food preservation, cosmetics, and medical applications. In addition, it is used in the manufacture of vaccines for the adsorption of protein on the surface of the mucosa, value-added product in carbohydrates and applied in different industrial products such as nutrition, biomedical, and prebiotics. In

A. Ebinesar · V. S. More (*) · D. L. Ramya · G. R. Amrutha Department of Biotechnology, Sapthagiri College of Engineering, Bangalore, India S. S. More School of Basic and Applied Sciences, Dayananda Sagar University, Bangalore, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_14

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this book chapter, we mainly focus on discussion about the application of chitosan in agriculture and industrial processes. Keywords

Fungi · Chitin · Chitosan · Bioremediation · Soil fertility

14.1

Introduction

Biopolymers are natural polymers that, along with their immunogenic properties, have the characteristic of being easily degraded by enzymes (Pokhrel et al. 2015). Different numbers of biological products, such as silk, alginate, elastin, chitin, and collagen, are known to be natural biopolymers. The advancement of natural biopolymers offers a hopeful direction for the development of research in a variety of fields such as agriculture, food, and treatment of medicinal and wastewater. (Correlo et al. 2005). Chitin is one of the commonly available biopolymers with a 1–4 glycosodic bond molecular complex and N-acetyl—D-glucosamine units that can be derived from many living organisms, such as insects, microbial cell walls, molluscs, and crustacean outer layers (Muzzarelli 1977). Chitosan is another biopolymer derived from the deacetylation of chitin biomolecule end up with formation of 1–4 glycosodic bond and glucosamine poly-carbohydrates molecular combination. These biopolymers are used in several industrial processes, such as the treatment of contaminated water: the remediation of metal ions, the flocculation and coagulation of protein and amino acid biomolecules and coloured dyes; in the food industry: the removal of suspended solids, the stabilisation agent for colour and food conservation and protection, the preservation agent, the gelling agent and the thickener; in the field of medicine: cholesterol management, bone fracture wound healing, inhibition of dental plaque, blood clotting agents and drug delivery; in the field of agriculture: seed coating, continuous release of agrochemicals and fertilisers, and also in the field of cosmetics: body lotions and creams, moisturisers (Davis 2011). Knorr (1991) reported that chitosan was isolated from crab shells and prawns over the decades by using alkalis under deacetylation at high temperatures. However, due to variations in the availability and cost of the above-mentioned raw materials, the output rate will not be constant (Crestini et al. 1996). In addition, the heterogeneous physical properties of the chitosan extracted from the crabs and prawns are shown. To solve these problems, through the fermentation process, the chitosan is effectively isolated from the farming of the respective fungi. Chitin has persisted with the cell surface of various fungi such as Ascomycetes septa, Deuteromycetes, Zygomycetes, Basidiomycetes to preserve their shape, the veracity of the cell structure and the strength of the cell wall (Ruiz-Herrera et al. 1992). The first-person isolated chitin from various forms of fungal species such as Agaricus acris, Agaricus cantharellus, Agaricus volvaceus, Hydnum hybridum, Hydnum repandum, and Boletus viscidus was Braconnot during the year 1811. In addition to that Odier (1823) isolated the chitin and chitosan successfully isolated from the

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Table 14.1 Comparison between different characteristic of chitin and chitosan biopolymers between fungal mycelium species and crustacean species Basic parameters Accessibility Inorganic constituents Method of extraction process Effectiveness of cost for waste management Pre-treatment Physicochemical properties Orientation of the plant immune activity

In fungal mycelia Easily available in all seasons and no topographical limitations Lower Very simple process Less

In crustacean sources Availability according to the seasons Higher Huge solvents are needed More

No need Remains same Very effective

Required Varied Less effective

insects crutile and from mushrooms. Novak et al. (2003) introduced the role and function of the group of amines and also explained the characteristics of the formation of chitosan. In the year 1894, Hoppe-Seyler (1894) was the first person proposed the name chitosan. White et al. (1979) is the first-person isolated chitosan from the fungal mycelium. Araki and Ito (1974) found catalytic mechanism involved for the bioconversion of chitin residue present in the cell wall into chitosan by the chitin deacetylase enzyme using Mucor rouxii fungal species. Very recently, the industrial processing of deacetylated chitin (chitosan) has been introduced, gaining more interest and being adopted by many countries. Compared with chitosan isolated from crustacean sources, the notable advantages of chitosan isolated from fungal mycelium were described (Knorr and Imeri 1989) by comparing different physiochemical parameters such as effect or degree of acetylation, dispersal of charged groups, molecular weight of chitin, and chitosan present on fungal chitosan, as shown in Table 14.1 (Adams 2004). In this book chapter, we discussed about the various physiological function of chitin and chitosan, synthesis from fungal species, application of chitosan and its derivatives in food industry, biomedical, cosmetics and agriculture.

14.2

Physiological Function of Fungal Chitin and Chitosan

The key constituents of neutral polysaccharides, chitin, glycoproteins and chitosan are the cell wall of the fungus mycelium, and the macro amounts of galactosamine polymers, melanin, fatty acids, and polyuronides (Wu 2004). In the cell wall of the hyphae and reproductive structure (spores), chitin biopolymers were observed conjugating with glucan molecules and forming microfibrils. In addition, to provide the rigidity and morphology of the cell wall, the microfibrils are enshrined as an unstructured matrix. The presence of chitin in certain fungal mycelium, such as Mucor, Rhizopus, and absidia, has been observed (Ruiz-Herrera et al. 1992), which contributes to the integrity of the cell wall and enhances the defence against foreign

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materials and also increases stability at high temperature (Adams 2004). Specht et al. (1996) depicted that the other functions of chitin and chitosan biopolymers compute by the effect of mutants bearing defect mechanisms in synthesis of chitin, accumulation of polysaccharides on cell walls and intracellular transferring in chitin synthases.

14.2.1 Biosynthesis of Chitin and Chitosan Biopolymers The synthesis of chitin is initiated by glycogen, the source material. The phosphorylase enzyme catalyses glycogen in this synthesis process and contributes to glucose1 phosphate conversion. Glucose 6 phosphate was further transformed by the enzyme phosphomutase and then by the obtained product into fructose 6 phosphate in the presence of hexokinase. And then, the process of amination and acetylation results in the transformation of Fructose 6 phosphate into N-acetyl glucosamine. In addition, isomerisation was performed by the addition of a 1, 6 carbon phosphate group catalysed by the enzyme phospho-N-acetyl glucosamine mutase. Additionally, the inter-conversion reaction followed to form uridine diphosphate N-acetyl glucosamine by the interaction of uridine triphosphate. Eventually, the converted UDP N-acetyl glucosamine formed chitin in the presence of chitin acetylase enzyme (EC 3.5.1.41) shown in Fig. 14.1 (Batista et al. 2018).

14.2.2 Fungi Used for the Isolation of Chitin and Chitosan In order to produce chitosan, Hu and colleagues studied various forms of fungal species. The ability to isolate chitosan is present in these isolated fungal strains. They found that the fungi, namely Basidiomycetes, Deuteromycetes, and Ascomycetes are not having native chitosan but the fungal zygomycetes comprised with chitosan fungi (Kirk et al. 2008) (Table 14.2). The quantity of chitin in fungal cell wall is precise to environmental factors, species, and age of the microbial sources. The percentage of chitin content specifically vary from 2 to 42% in the Euascomycetes, fungal, and yeast.

14.2.3 Factors Distressing Fungal Chitin and Chitosan Conversion Campos-Takaki et al. 1983 suggested that due to environmental factors, substrate conditions and intrinsic characteristics of fungal organisms, the amount of chitin and chitosan biopolymer found on the cell of the fungi differs. According to the mechanisms involved in the biosynthesis process in fungal cells, they found the impact of different factor affects chitosan formation by using submerged fermentation process. These variables are primarily concerned with enhancing the synthesis of chitin and also optimising the physiochemical characteristics (Tanaka 2001). Additionally, these features improved the metabolic activity of fungi followed by

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Fungal Chitosan: The Importance and Beneficiation of this Biopolymer in. . .

Glycogen

Glycogen phosphorylase

Glucose 1 Phosphate Glucose 6 Phosphate Glutamine Glutamic acid Acetyl CoA CoA

UDP PPi

315

Fructose 6 Phosphate

Phosphoglucomutase

Glucose 6 isomerase Glutamine 6 P aminotransferase

Glucosamine 6 P Glucosamine 6 P N acetyltransferase N acetyl glucosamine 6 P

Phospho –N-acetyl glucomutase

N acetyl glucosamine 1 P

uridine diphosphate Nacetyl glucosamine P Chitin

Fig. 14.1 Schematic representation of synthesis of chitin

catabolic repression (Denuziere et al. 2001). Researchers have found that, by adding different nutrients, the growth of fungal mycelium and the formation of biopolymers can be enhanced. Nwe and Stevens (2004) observed a substantial uplift of total chitosan generation in the Gongronella butleri fungal species by the addition of urea. The added urea serves as a source of nitrogen and helps increase the substantial elevation of biopolymer molecular weight in the two quantities of chitosan. Mishra and Kumar (2007) investigated the efficacy of chitosan production by using different sources of nitrogen such as yeast extract, dry cyanobacterial biomass, urea, Pleurotus ostreatus, and ammonium sulphate and they found the huge amount of biopolymer collected in the occurrence of cyanobacterial biomass because of its nutritional value. An experiment was performed by Benjamin and Pandey (1997) and his colleagues to investigate the production of biopolymers using coconut oil cake as a substrate for the production of chitosan under well-optimised conditions in candida rugosa. In addition, the influence of different sources of carbon such as sucrose, pectin, glucose, and galacturonic acid on the growth of Aspergillus niger fungal biomass was examined by submerged fermentation processes (Solís-Pereira et al. 1993). Finally, they observed that the complementary carbon nutrients induce to increase the biomass amount. Xie and West (2009) showed that the high acidic pH action of the solid substrate helps to increase the biomass content by a large amount.

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Table 14.2 List of fungi used in the production of fungi by various methods Fungal strain Rhizopus oryzae Mucor rouxii/ SMF A. glauca M. rouxii

G. butleri USDB 0201 Mucor racemosus Cunninghamella elegans P. blakesleeanus Gongronella butleri R. oryzae

R. oryzae TISTR 3189/SMF G. butleri IF08081 A. coerulea/SMF

Chitosan yield 4.3 g/kg of soya bean residue 0.61 g/L for molasses salt medium 7:4% of dry cell weight 7.3% of dry mycelial weight—Synthetic medium 4–6 g/100 g mycelia—Sweet potato 35:1 mg/g dry mycelia

Temperature 25–45  C

Fermentation process SSF

25–45  C

SMF

25–45  C

SMF

25–45  C

SMF

Synowiecki et al. (1997)

25–45  C

SSF

Nwe et al. (2002)

25–45  C

SMF

20:5 mg/ g dry mycelia 10:1 6 2:9% dry mycelia 1.19 g/L of chitosan per litre of apple pomace extract 1.13 6 0.10 g/L of whey medium (in the presence of gibberellic acid) 138 mg/g or 14% of dry mycelial weight— PDB medium 730 mg/L of Shochu distillery effluent 4.11 6 0.08 g/L of synthetic medium

25  C45  C

SMF

25–45  C

SMF

25–45  C

SMF

Knorr and Teutonico (1986) Da Silva et al. (2001) Da Silva et al. (2001) Streit et al. (2009)

25–45  C

SMF

Chatterjee et al. (2008)

25  C45  C

SMF

25–45  C

SMF

25–45  C

SMF

Pochanavanich and Suntornsuk (2002) Yokoi et al. (1998) Mélida et al. (2015)

References Suntornsuk et al. (2002) Streit et al. (2009) Hu et al. (2004)

Chatterjee et al. (2008) optimised the production of chitosan biopolymer in M. rouxii and R. oryzae in the presence of salt treated molasses and whey growth medium and the effect of auxins, gibberellic acid, and kinetin. He found that, in the presence of low plant hormone concentrations, the chitosan content increased in both fungal species and, in the case of higher plant hormone concentrations, the growth of both microbial species was decreased. Some research studies depicted that the amount of biopolymer separated from the fungal species which harvest at their log phase.

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14.3

317

Application of Chitosan in Food Industries

Because of its physiochemical parameters such as molecular weight (2–200 kDa), chitosan is used in various disciplines such as food science, medicine, agriculture, and cosmetics to break down the acetyl compound from chitin based on the solubility of the polymer chain in the presence of weak or strong acid. The solubility of chitin was improved by the protonation of the D-glucosamine amine group and eventually converted under acidic conditions to polysaccharides. Due to its biological activity and its protection for human beings, the comprehensive application of chitosan would be more favoured. In food industries, the application of chitosan has more fascinated consideration as a natural preservative because its antimicrobial activity in contradiction of different foodborne microbes like fungi, bacteria, and yeast. Due to the electrostatic interaction between the cationic charged biological ligand and the anionic charged microbial cell surface ligand, this biopolymer attempts to adjust cell diffusivity. The degradation of protein and other cellular compounds (Young et al. 1982) is accomplished by this interaction. In addition, by avoiding the diffusion of hydrolysed mRNA products and the synthesis of protein compounds, metal complexation, spore forming elements and nutrients, this biopolymer contributes to the antimicrobial mechanism (Hadwiger and Loschke 1981). Uchida et al. (1989) reported that chitosan has an important antimicrobial activity to inhibit fungal and bacterial growth in contrast to chitosan oligomers. In addition, the biopolymer’s antimicrobial effect also depends on its molecular weight, degree of deacetylation, and type of bacterium (Tsai et al. 2005). The loss and gain of microbial growth inhibition by mixing chitosan into various food compounds containing carbohydrates, fat, protein, salts, and vitamins was studied by Roller and Covill (1999). In addition, Devlieghere et al. (2004) found that there was a negative effect on the addition of salt, starch, oil, and proteins to prevent the growth of microbial flora.

14.3.1 Effect of Chitosan in Bread Bread, the most disbursed food mainly made from wheat flour used worldwide. Martins et al. (2017) suggested that the synergistic impact on gluten molecules such as intolerance, allergy, and celiac disease is a major challenge for researchers to explain the alternative source of bread output. Starch is a raw material component that can be found in various sources, such as maize, potatoes, rice, and cassava (Ferreira 2019). In addition, the pigment rice has higher levels of starch, protein, vitamin B, and minerals and consists of high nutritional values of pro-anthocyanides, carotenoids, and anthocyanins. Garry Kerch (2015) studied staling phenomena in order to verify the consistency of the bread’s flavour and texture with regard to time. By hindering the initial toughness of a starch gel and limiting microbial growth, this blending of chitosan increases the shelf-life span. The research to increase the shelf life of baguette by impregnating chitosan with a molecular weight of 493 kDa was conducted by Park and his co-workers. The efficacy of the addition of chitosan was

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tested by coating various chitosan concentrations on the surface of the dough in the range of 0.5–1.5%. He noted that due to the moisture-resistant property of chitosan, 1% of the chitosan-coated sample showed many characteristics such as hardness, less weight loss, and retrogradation than the uncoated baguette at 26  C for 36 h (Butler et al. 1996). The chitosan creates a barrier layer on the dough surface and resists the presence of moisture on the bread surface, thereby reducing weight loss, retaining hardness, retro deterioration, and relative to the regulation, the shelf life also increased one more day. Similarly, Park and Chong (2002) detected that the shelf life of bread can also extend to 24 h by coating the chitosan that has the molecular weight of kDa. Not only does the chitosan biopolymer boost retrogradation, but it can also increase the shelf life and consistency of the bread by inhibiting the growth of the microbes and preserving the property of antioxidation. Similarly, with a molecular weight of 120 kDa combined with 0.3% lactic acid, the chitosan concentration increased from 1 to 2%, decreasing the bacterial count and thiocomplexed barbituric acid reactive compounds and increasing the water content after 8 days at room temperature compared to uncoated chitosan. The presence of mould was found after 4 days in the control. Lee et al. (2002) revealed that the carboxymethyl modified chitosan also increases the shelf-life period of fermented bread by inhibiting the microbial growth and delaying interaction between amylose and amylopectin in starch molecule. Similarly, the same research group examined the antimicrobial activity of chitosan by varying the molecular weight from 1, 5, 30, and 120 kDa with a degree of deacetylation of lower (Mw 1 and 5) and higher molecular weight chitosan was (30 and 120 kDa) 92% and 85%, respectively, and also in the presence of lactic acid and reacted with varying dough concentrations 0.01%, 0.1%, 0.3%, and 0.5%, respectively. They found that, after 8 days of incubation at room temperature, the lower concentration of chitosan and control showed similar results in bacterial growth of about 106 CFU/mL viable cells. Colour, water content, pH, and titratable acidity analysed by (Silva et al. 2020), basic volume and textural properties of gluten off bread by reaction between red rice flour starch and cassava flour and transglutaminase enzyme with a chitosan concentration of 0, 1, and 2%. The presence of chitosan and the activity of the transglutaminase enzyme alter the bread structure and the enzyme and chitosan increase the stabilised network. Although the presence of chitosan may also have some unfavourable consequences, it decreases the lag time of yeast fermentation follow-on in bread with less precise volume and greater inflexibility and chewiness.

14.3.2 Effect of Chitosan in Fruits and Vegetables In general, due to the lack of proper storage devices, fruits and vegetables are contaminated by yeast, fungi, bacteria, and mould after harvesting (El Ghaouth et al. 1991). By edible coating and storage of fruits and vegetables in cold temperatures, Park et al. (2005) attempted to minimise microbial infection. The non-toxic coating serves as a protective shield, decreasing the rate of fruit respiration and transpiration, not allowing microbial species to develop and change in colour,

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increasing the quality of texture (Kester and Fennema 1986). By altering the levels of ethylene, oxygen, and carbon di oxide, the semipermeable membrane shown by the coating keeps back the ripening of the fruit. In addition, the chitosan coating avoids the diffusion of levels of carbon di oxide as compared to oxygen in order to avoid the growth of anaerobic microbes in the inner atmosphere of fruit and vegetable tissue and to extend the storage time and to regulate the fruit deterioration. El Ghaouth et al. (1991) analysed the antimicrobial activity on the surface of strawberry fruit by coating chitosan biopolymer in the presence of Botrytis cinerea and Rhizopus sp. As a result, the decay of the fruit and the consistency of the fruit are decreased by the subsequent dip of the fruit into chitosan solution (1 and 1.5%) at 13  C. The chitosan forms a powerful barrier, prolongs the time for anthocyanin synthesis and high acidity, decreases the rate of growth and breathing of fungicides (Reddy et al. 2000). El Ghaouth et al. (1991, 1992) delineated that the biopolymer has the capability to inhibit the growth of fungal by persuading the chitinase enzyme to chitosan has the ability to inhibit growth of several fungi to induce chitinase, a defence enzyme, and to provoke phytoalexin in pea shells. Enzyme-induced defence mechanism by the combination of chitinase and β-1,3-glucanase. Notably, the enzymatic defence mechanism is not so effective in the presence of chitosan in unbroken fruit. This is mainly because of the intact fruit surface that does not allow the surface of chitosan and tissue to interact. In strawberry cuticles that have no porus on the surface, a similar form of observation was made. The edible coatings have recently been used as a method for combining different functional components such as flavours, chromophores, nutraceuticals, and antimicrobials agents (Han et al. 2004; Hernández-Muñoz et al. 2006). Calcium, potassium, vitamin E, and oleic acid are combined with chitosan and used as an edible coating to improve the shelf life and nutritional value of fruits and vegetables. The capacity of antimicrobial activity and moisture barrier, weight loss, colour on strawberries and red raspberries were demarcated by Han et al. (2004), by the three distinct chitosans such as native chitosan, 5% glucanol modified chitosan and 0.2% DL-alpha-tocopheryl acetate modified chitosan. This three distinct chitosan has a deacetylation degree of about 87% and is treated as 1 and 2% with separate acetic acid concentrations and incubated at lower temperatures. The results obtained have shown that the chitosan coatings selected are capable of increasing antimicrobial activity, reducing the rate of colour changes and decreasing pH changes. The addition of calcium and vitamin E, however, results in substantial improvements in weight loss and regulation of moisture. Due to the hydrophilic aspect of the chitosan films, the calcium and vitamin E modified chitosan decreases the penetration of water molecules through the matrix (Amarante and Banks 2001).

14.3.3 Effect of Chitosan in Kimchi Kimchi, a mixture of Chinese cabbage and various ingredients, such as red pepper, green onion, garlic, and fermented fish sauce (Choi et al. 2016), is a fermented

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vegetarian food. No et al. (1995) elucidated the edible period of the kimchi food can extensively increase by the addition of chitosan oligosaccharides with the concentrations of 0.05–0.02% in 0.2% of water. They reported that, relative to the control at 20  C, the addition of chitosan increased the shelf-life span from 2 to 6 times (Yoo et al. 1998). No et al. (1995) evidenced that the period of deterioration can be extended for 10 days by salt-modified chitosan (0.1–0.15%) comparatively to control. Chitosan biopolymer decreases the softening of cabbage tissues and the interaction of chemical compounds. In addition, chitosan blended in preservatives, medical herb extracts, sodium benzoate, liquid calcium form which helps to improve quality and minimise degrading effects (Son et al. 1996).

14.3.4 Effect of Chitosan in Meat In general, meat has the property of being quickly affected by lipid oxidation, contributing to rancidity on the surface of the meat. Chitosan serves as an antioxidant and antimicrobial ability to keep back lipid oxidation and hinder the growth of microbes that spoil food on the meat surface, as shown by several researchers. Darmadji and Izumimoto (1994a) showed that the addition of 1% of chitosan decreases the value of thio-barbituric acid (TBA) on the meat surface by 70% compared to the control stored at 4  C for 3 days. The value of TBA remains same for 10 days in the presence of 0.5–1.0% of chitosan. Likewise, chitosan has the property to enhance the colour of beef at the time of storage. Darmadji and Izumimoto (1994b) analysed the stability of the storage of minced beef blended with 100 ppm of nitrite and Lactobacillus plantarum with chitosan cumulated with nitrite and Lactobacillus plantarum. He noticed that bacterial growth decreased to double the log cycle, TBA decreased to 36%, and the remaining nitrite degenerated by 63%, creating a better colour on the fermented surface. The growth of bacterial cells inhibited on chilled pork by the addition of 0.3–0.6% of chitosan glutamate to immature minced pork was investigated by Sagoo et al. (2002). He found that the chitosan reduces the count of viable cells, lactic acid bacteria by 3 log of CFU/g yeast, and moulds for 18 days at 4  C. Juneja et al. (2006) depicted that the additional 3% of chitosan glutamate which has deacetylation of around 86% significant effect on reducing Clostridium perfringens spore at 7.2  C in 12 and 15 h. In addition, the shelf life of the pork can be increased from 0.1 to 1.0% by the different chitosan concentration, which has a molecular weight of 30 and 120 kDa. The dipping of the fork into the chitosan biopolymer was observed, and 5% trisodium phosphate decreased the number of aerobic bacteria on the surface of the pork. Wu et al. (2000) disclosed that soy protein did not have a major effect on reducing or regulating lipid oxidation when packing pork with chitosan (DD-85%: in the presence of 1 and 2% formic acid). Because of that, on the beef surface, the existence of TBA increased, leading to increased oxygen permeability.

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14.3.5 Effect of Chitosan Added with Seafood and Seafood Products Due to lipid oxidation of fatty acids, the mechanism catalysed by the presence of hematin compounds and metal ions in the fish muscle (Decker and Hultin 1992) can possibly destroy seafood. In addition, the consistency of seafood deteriorated by autolysis, protein loss, and contamination demonstrated by microbe development (Jeon et al. 2002). Kamil et al. (2002) investigated the antioxidant activity of seafood with different viscosities in the range of 14–360 cP, molecular weight of chitosan 660–1800 kDa in cooked, comminuted flesh. Chitosan concentrations of 50–200 ppm and traditional antioxidants such as tert-butylhydroquinone, butylated hydroxy anisole, and butylated hydroxytoluene were evaluated for the effect of oxidative stability and maintained at 4  C. From the obtained result, they observed that optimised parameters for reducing lipid oxidation on surface at viscosity 14 cP, chitosan concentration at 200 ppm for 8 days stored at 4  C. It clearly reported that the antioxidant potential on the fish surface was obtained at the molecular weight of chitosan and at its concentration of 200 ppm and 14 cP viscosity. Peng et al. (1998) showed that chitosan modified by ferrous ions decreased lipid oxidation on the surface of the fish. Amino groups have been active in metal ion chelation.

14.4

Applications of Chitosan in Pharmaceutical and Biomedical Field

Chitosan is used in pharmaceutical formulation and drug delivery in order to increase sustained release of various drugs–vaccines, antibiotics, proteins, antiinflammatory drugs, peptides, factor of growth, antimicrobial therapies, gene therapy, and delivery of genes. In addition to that, this polymer used in the area of tissue engineering for the regeneration of wounds and burns, tissue, bones, neurons, muscles, cartilage, tendons, liver, ligaments, and also applicable in different diagnostic approaches in skin treatment, cancer therapy, ophthalmology, and dental care. Moreover, the chitosan has the property of sensing heavy metals, bioimaging, matrix for holding the enzymes and in the field of veterinary medicines.

14.4.1 Oral Sources of Dosage Sawayanagi, Y investigated the extent of disintegrating of oral dosage by the blending of chitin, chitosan or MCC and mixture of lactose or. From the result he observed that the chitosan has more potential on sustained drug release with respect to time function which is shown in Fig. 14.2 (Sawayanagi et al. 1982; Ofori-Kwakye and Fell 2001). The lag time of disintegration potential of the three biopolymers additives are arranged in the increasing order (chitosan > chitin > MCC) with respect to their respective concentrations. It is mainly due to the angle of rest and it was considerably decreased by enhancing the concentration of chitin or chitosan biopolymers mixed with lactose or potato starch comparatively to the MCC. Chitin

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Fig. 14.2 Chemical constituents of chitin and chitosan biopolymers (Kato et al. 2003)

and chitosan are extensively applied in the place of MCC due to their higher lubricating property. Likewise, the complexation of chitosan and lactose increases the tablet hardness with respect to rise in chitosan concentration, suggesting that chitosan also has a strong combinative property than MCC (Sawayanagi et al. 1982). The important additives for direct compression have been reported to be chitin and its derivative, Chitosan.

14.4.2 Dressing of Wounds To accelerate the early recovery, lesion dressing is essential for burn wound healing. The purpose of bandaging is to reduce the development of hematoma and inflammation, to limit dead space, to defend against further infection or trauma, to retain drainage, to create sufficient oxygen stress, to maintain a humid atmosphere, and to minimise movement (Choate 1994). A moist condition that facilitates angiogenesis is important for the distribution of wound healing cellular components (Liptak 1997). The effective wound dressing will have the following characteristics: (1) avoidance of growth of bacteria on the injured skin, (2) exudate captivation, and (3) epidermal formation and granulation. Currently, the Beschitin® -W (Unitika Ltd., Osaka, Japan) introduced the chitin in the bandage as a protective coverage material for the wound healings. The therapeutic effects were studied using a rat thermal damage model by the four different forms of wound dressings which include three films and one spongy membrane, i.e. chitin/chitosan mixture spongy membrane, chitin/chitosan mixture film, chitin film, and chitosan film (Tachihara et al. 1997a, b). Directly after drenched in the buffer, the chitin/chitosan spongy membrane fascinated some solvent, and then incremental water absorption was observed,

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indicating that higher absorption was observed in the spongy membrane compared to any other films examined. Similarly, in comparison with the other film forms, the spongy membrane also showed high silver sulfadiazine (SSD) content. The spongy membrane believes to have more microscopic structure in turn which has the ability for high drug concentration.

14.4.3 Muco-Adhesive Oral Mucus, viscoelastic gel adhered to tissue of the mucosal region in various organs such as respiratory, reproductive tracts, eyes and gastrointestinal in human body. It comprised with H2O (90–98%), 0.25–5% w/w of mucins, salinity level 0.5–1%, percentage of protein 0.5% w/v, DNA, bacteria, lipids, and cellular debris (Khutoryanskiy 2011). Mucins is the major component of the mucus; it has gel-like property due to the presence of glycoprotein. The glycoprotein has protein core, and it is covalently bonded with the side chain of its carbohydrate molecule through O-glycosidic linkages (Peppas and Buri 1985). Several research articles stated that the chitosan derivative has the muco-adhesive properties. The hydroxyl and amine functional group of chitosan has the characteristic of increasing the solubility of chitosan at lower pH conditions. In acidic condition, the pronated amine group electrostatically interact with the sialic acid and the epithelial surface. Importantly, the muco-adhesion characteristic formed by the hydrogen bond and apolar interactions. Recently, chitosan derivatives used as a tool for transmucosal drug delivery process in the discipline of nasal vaccination and eye drops. The commercial oral muco-adhesive film was initially introduced by WaplonP® (Kowa Co., Ltd., Aichi, Japan) for the treatment of stomatitis. In this product, the mucoadhesive property brought by the combination of chitosan and sodium alginate. Kawasaki stated that modified chitosan helps for the sustained release of and diltiazem which shows the efficiency more than two times than oral administration (Kawase et al. 1997).

14.4.4 Adhesive for Water Resistance Naturally, the marine animals used water-resistant protein to attach on the submerged surfaces. The protein biomolecules built with repetitive groups of hydroxyproline, dihydroxyphenyl aniline, and lysine. The adhesion process carried out by conversion of o-diphenolic residues to O-quinone mediated by catechol oxidase enzyme (Waite and Tanzer 1981). Then the O-quinone followed a non-enzymatic reaction to make complex with lysine protein molecules to form protein gels which give more adhesive strengths. But there is no well-defined literature proof for the interactions so far. Yu and Hwang (1999) reported that the oxidation of dihydroxyphenyl aniline has the capability of gel formation but the non-oxidative form of DOPA can be used as a water-resistant adhesion material. Waite stated that the mechanism involved in the water-resistant adhesion by the

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quinone chemistry is very complex. In addition to that Conner and Hemingway (1989) narrated three different roles in the adhesion process by the phenols and quinones that are very remarkable. The three steps are (1) the phenolic residues present in phenol formaldehyde is having similar structure with the polyphenolic adhesive proteins, (2) the synthetic quinone comprised polymeric compounds has adhesive properties, (3) dihydroxyphenyl aniline converted to O-quinones by autooxidation process. Domard (1987) used to tyrosinase enzyme oxidise the low molecular weight compound dopamine and then the quinone reactions facilitated by the biopolymer (Chitosan) comprised with polysaccharides, and it is not a protein. The amine group present in the chitosan deprotonated at dissociation constant 6–6.5. Muzzarelli et al. (2001) reported that the adaptation of phenolic on chitosan mediated by tyrosinase converted into quinone-tan modified chitosan, formed crosslinked gel. In earlier studies, the same researchers revealed that the quinonetanned chitosan behave like ag gels which has water-resistant adhesive properties.

14.5

Carriers for Drugs

14.5.1 Microparticles/Nanoparticles Studies performed, gadolinium neutron capture for cancer treatment mediated by chitosan nanoparticles acts like a chitosan nanoparticle act like drug carrier for gadolinium (Yamada et al. 2000). In this study, the drug gadolinium was administered concentration in tumour tissue on mice around 74% and delivered with the help of gadopentetic acid-adapted chitosan nanoparticles which is approximately 430 nm. The result revealed that the tumour growth was effectively repressed by chitosan-modified gadolinium. Chitosan colloidal particles can be successfully used as an oral vaccine due to its quick absorption on lymphoid tissue (Tokumitsu et al. 2000). Similarly, the interaction of (IgG) immunoglobin and local (IgA) with DT-adapted chitosan shows a noteworthy effect comparatively than intragastrical alimentation with diphtheria toxoid (DT) in PBS (Van der Lubben et al. 2000). From the safety point of view, non-viral gene transmission has gained a lot of attention. In addition to that, several studies have reported that the different types of cell lines could be transfected with the use of DNA-nanospheres (200–750 nm) mediated by gelatin and chitosan (Van der Lubben et al. 2001). But the DNA-chitosan nanospheres could not be crosslinked during the preparation. While DNA-chitosan nanospherical particles had relatively low in vitro transfection efficiency and were close to DNA-gelatin nanospheres, many non-viral vectors suffer from the problem. During the fabrication of nanoparticles, different types of water loving and water hating drugs loaded with chitosan nanoparticles. The loading efficacy of the drug mainly depends upon the method of separation and physiochemical properties. In cancer therapy process, the polar 5-flurouracil completely adhered with chitosan nanoparticles (200–300 nm) chemically crosslinked in the presence of glutaraldehyde in water in oil emulsion process. Mitra (2001) described about the suppression of tumour by conjugating the doxorubicin drug with chitosan nanoparticle using

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reverse micelle process (Van der Lubben et al. 2003). Janes et al. (2001) reported that tripolyphosphate and crosslinked chitosan nanoparticles is used to deliver lots of small molecular drugs and bio-macromolecular treatment. For example, through ionotropic gelation of the chitosan with tripolyphosphate, researchers effectively encapsulated DOX with the chitosan nanoparticles. The cytotoxicity of DOX-binded nanoparticles in A375 cells of human lump and C26 cells of murine colorectal carcinoma showed that nanoparticle dextran sulphate-containing formulations were able to slow cytostatic action relative to unmodified DOX.

14.5.2 Conjugates The functional groups such as amine and carboxylic group present in N-succinyl complexed chitosan (Suc-Chi) derivatives can easily react with several compounds shown in Fig. 14.2. The conjugation of (Suc-Chi) with water-soluble carbodiimide (EDC) was mediated by direct link of mitomycin C (MMC) and changed in waterinsoluble compound and water-soluble conjugates (Leong et al. 1998). After administration, the distribution of (Suc-Chi) in different organs such as liver, kidney, and spleen is less than 5% of dose per gram of tissue (Mitra et al. 2001). Moreover, these conjugates with less than 10% of the dose/g tissue administered and tested in preputial gland, small intestine, backbone, femoral muscle, and peritoneum organs. But more than 10% of N-succinyl-modified chitosan was transferred to the prostate and lymph nodes for treatments (Janes et al. 2001). The conjugates can increase the molecular weight, and it lowers the biodegradability when it is complexed with succinyl groups. The anionic charges help extend the half-life of the polymers in the systematic circulation process due to minimum interaction with tissues and blood vessels (Song et al. 1992) and supermolecules are described to stay longer in the body related with low molecular weight molecules (Kato et al. 2000a, b).

14.5.3 Antitumor Activity Hydrophilic Suc-Chi-mitomycin C conjugate, antitumor activity was examined in 180-, M5076-, and MH134-bearing sarcoma mice (Kato et al. 2003). Such conjugates show strong antitumor activity. Suc-Chi-MMC conjugates (Suc-ChiMMC conjugates) were also shown to have strong antitumor activity contrary to sarcoma 180, melanoma of B16, and murine leukaemia (L1210 and P388) (Takakura et al. 1987; Sezaki and Hashida 1984). Although the conjugate compounds are much better, with a slight reduce in body weight but no lethality, but the excess quantity of free drug subsidised to lethal side effects. The anticancer activity of MMC decreased by the chemical modification of the aziridine ring, one of the active groups when the substituents is increased (Kato et al. 2002a, b).

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14.5.4 Enhancers for Intestinal Absorption N-trimethyl-chitosan acts as an ampholyte ion consisting of quaternary amines by methylation of amino groups and mono-N-carboxymethyl-chitosan (mCM-Chi) shown in Fig. 14.2. As intestinal absorption enhancers, these chitosan derivatives are productive because they open tight intracellular junctions through interaction with mucosal epithelial cells.

14.5.5 Tissue Engineering Researchers have stated that conjugates of chitosan and thioglycolic acid used as an innovative material for the scaffold process. In its structure, Chi-TGA has immobilised HS groups (Fig. 14.3), and the formation of disulphide bonds with the polymeric system was revealed by the conjugate process. The e application of sulphide groups with chitosan forms a new polymer showing dissimilar properties (water solubility, biodegradability, and muco-adhesion, in situ gelling). At a physiological pH and temperature (at 37  C), the gel-like scaffolds organised using Chi-TGA conjugates can form a regular shape parallel to the native tissue because in situ gelling depends on the quantity of thiol groups encapsulated to the polymer at the concerned pH.

Fig. 14.3 Application of chitin and chitosan derivatives

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14.5.6 Dentistry The application of chitosan in dental medicine has the properties of bioactivity, wound healing, anti-inflammatory, bone repair, and hemostatic actions (Shirahata 1984; Hamman et al. 2003). The different forms of chitosan are used as a solution form (acetic or methane sulphonic acid used as solvents) in the form of microspheres, hydrogel and toothpastes, and its combination with additives such as amorphous calcium phosphate, amelogenin, and quinic acid has enhanced the ability of these chitosan preparations to prevent tooth decay and enamel degradation. The gel/hydrogel form of chitosan refers to the treatment of chronic periodontitis and canker sores. Chitosan can play an important role in preventive dentistry because of its inherent flexibility, efficacy, and the ability to act as a protective shield to the intrusion of acids into enamel and its mineral loss. Antimicrobial effect on oral biofilm and decrease in the number of Streptococcus mutans in the oral cavity are functions of toothpastes, mouthwashes, and chewing gums dependent on chitosan and herbal fullness. Chitosan complexes and microparticles of fluoride improve the absorption and safety of fluoride cavities. Chitosan-based endodontic cements minimise inflammation and help the rebuilding of bone.

14.5.7 Veterinary Medicine In veterinary medicine, researchers investigated the applications of chitosan curing of wound, regeneration of bone, antimicrobial and analgesic effects (Queiroz et al. 2015). The application of chitosan for drug and vaccine delivery in veterinary animals and as a dietary element was also addressed. The chemotherapeutics such as painkillers, antiparasitics, anaesthetics, antibiotics, and growth promoters to mucosal epithelium for absorption for local or systemic operation are the veterinary drug delivery, areas most likely to benefit from chitosan and the delivery of immunomodulatory agents to the lymphoid tissue associated with the mucosa for local immune response activation or modulation.

14.5.8 Cosmetics Generally, most of the chitosan products has huge molecular weight which is very difficult to enter into the skin and makes a significant effect in skin care. The major chitosan derivatives used for the skin care products are chitosan complexed hydrochloride, acetate, lactate, carboxymethyl, oligosaccharides, as well as chitin sulphate, carboxymethyl chitin, and quaternised derivatives. It can be used on both dissolved and solid forms in aqueous solutions. The chitosan-blended cationic cosmetics are bacteriostatic, fungistatic, film-forming, antistatic moist-retaining and retains the moisture content very less and preserve the hair style with high humidity and also it releases the bioactive compounds very slowly. The chitosan biopolymer is more companionable with the components such as starch, glucose,

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Table 14.3 Applications of chitosan and its oligosaccharides in dentistry (Queiroz et al. 2015) Topic Surgery in dental Dental treatment Dental constituents Transplants Drug delivery systems Oral hygiene Antibacterial effect Healing dentistry Dental composites Wound healing Nano-dentistry

Form Solution Hydrogel

Application Periodontitis, erosive lesion, dental plaque inhibitor, agent to prevent dental caries Constituent in dentifrices

Toothpastes

Dental care: Toothpaste, chewing gum, Oral care

Bioadhesive Powder

Increase salivary secretion, antibacterial effects Fluoride delivery

Granule Nanoparticle

Acts as adhesives for dental Nano-biomaterials

Sponge

Used to enhance wound therapeutic in bone tissue

Composites

Use for scaffolds and transporters for molecular treatment Cell-defensive action Cell shielding action

polyols, oils, fats, saccharose, acids, nonionic emulsifiers, waxes, and nonionic water-soluble gums. Additionally, this biopolymer used in hydrating agents, solar filters, and other bioactive materials. Some of the applications for chitosan (Table 14.3) are: hair care, e.g. shampoos, colouring products, hair spray, and setting lotion, creams, and lotions (products for the face, hand, and body), colour cosmetics (make-up, nail polish, eye shadow, and lipstick), deodorising products, active agent microencapsulation, and dental care, e.g. toothpaste, tooth gel, and mouthwash (Queiroz et al. 2015). Chitin is also beneficial in cosmetology since the skin tolerates it well. It is an efficient moisturising agent and a film-forming tensor that often has two advantages: it provides water and prevents dehydration. Chitosan and chitin also have metal-chelating properties that are responsible for a very large number of exposure allergies. There are several commercially available products based on chitosan for cosmetic use: Curasan™, Hydamer™, Zenvivo™, Ritachitosan®, Chitosan MM222, etc (Tables 14.4 and 14.5).

14.5.9 Antimicrobial Agent The antimicrobial effect of chitosan leads by the electrostatic interaction between chitosan and the functional groups present on the surface of the bacteria and damage the physiological membrane, e.g. by facilitating the leakage of intracellular components and also by suppressing the flow of nutrients into the cells. Chitosan’s

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Table 14.4 Applications of chitosan and its oligosaccharides in veterinary medicine (Queiroz et al. 2015) Topic Types of drug delivery systems Delivery of vaccines Accessory Properties of biological effect Immunisation of mucosal Healing of wound

Form Solution

Application Sustained release of drugs in animals

Powder Microsphere Microcapsule

Mucosal formations Mucosal delivery of antigens Enhance the immune response Haemostatic Wound healing action

Regeneration of tissues

Nanoparticle Gel, hydrogel Sponge

Nutritional supplement Nanomedicine

Film Filament

Regenerative medicine in the field of tissue engineering Vaccines Surgical threads

Table 14.5 Applications of chitosan and its oligosaccharides in cosmetics (Queiroz et al. 2015) Topic Toiletry Hair care Personal care Cosmeceuticals Oral care Dental care Hygiene Skin care Essential oils

Form Solution Powder Film

Application Functional additives Moisturisers: Maintain skin moisture, tone skin Thickening agent Hydrating and film-forming agent Role in surfactant stability; stabilise emulsion Antistatic effect Bacteriostatic Delivery systems Products: Shampoos, creams, skin creams, creams for acne treatment, lotions, bath lotions, nail polish, fixtures, make-up powder, lacquers, nail lacquers, nail enamel, varnishes, hair sprays, hair colourants, wave agents

antimicrobial effect against a spectrum of microbial and fungi arising from its polycationic existence is well known.

14.5.10 Anticholesterolaemic Effect It is known that the entrapment instigated by a viscid polysaccharide solution decreases the solubility and absorption of dietary fatty acids. Conversely, the electrostatic force of interaction between chitosan and anion substances such as bile acids and fatty acids is measured by the involvement of the amino group in its structure. The interaction of chitosan with anionic surface-active materials depends on its three reactive functional groups: the secondary amino group, C-3 and C-6 primary and secondary hydroxyl groups, respectively. While considerable efforts

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have been made to find a connection between the physicochemical characteristics of chitosan and its ability to bind fat, only a few important associations have been shown (Morin-Crini et al. 2019).

14.5.11 Antioxidative Activity Chitosan has substantial scavenging ability against various free radical species, which gives similar effect that caused with commercial antioxidants. The existence of toxic-free radical will be scavenged by the chitosan derivatives such as 1.1diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide, hydroxyl, and alkyl radical samples isolated from crab shell with DD (degree of deacetylation) of about 90%, 75%, and 50%, respectively. The obtained results were showed that the higher DD chitosan biopolymer exhibited the highest scavenging activity against the free radicals. Chitosan Mw was found to have a negative association with activity. The chitosan sulphate derivatives had a higher scavenging effect on peroxide radicals, but the lowest Mw chitosan demonstrated a higher ferrous ion-chelating potential versus others (Chen and Chou 2005). The major reason for selecting chitosan as a natural antioxidant for stabilising the lipid containing foods and also to extend their shelf life by the chelation of metal ions. By chelating ferrous ions existing in the system, chitosans can retard lipid oxidation, thereby eliminating their pro-oxidant activity or their convert to ferric ions.

14.6

Applications of Chitosan in Agriculture

Generally, the plant pathogens are most economical species used as agricultural microbial source around the world. The pathogens are mainly controlled by chemical pesticides. But the usage of chemical pesticides leads two major issues (1) propagation of confrontation in pest inhabitants and (2) increasing the contamination of unpreserved agricultural crops. Several environmentally friendly approaches have been used to reduce the harmful effects of synthetic pesticides in order to solve these problems. These methods are capable of reducing the concentration of pesticides but these processes are not economically feasible (Chen and Chou 2005). Subsequently, to minimise the effect of pesticides, certain traditional approaches are used, but these techniques are not economically viable and are very difficult to apply in practical circumstances. So, an alternative that is very healthy and eliminates the harmful effects on human health, and the environment is introduced by natural polymer chitosan (Lim and Hudson 2004).

14.6.1 Chitosan in Managing Plant Diseases The chitosan biopolymer can be extensively used to monitor the pathogens occurring in the plants and the successive of the application of the polymer depends upon the

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Fig. 14.4 Some projected properties of chitosan act as elicitor of plant defence mechanism (Orzali et al. 2017)

pathosystem, chitosan derivatives, concentration of the biopolymer, degree of deacetylation of chitosan, viscosity, and effect of treatment (Jeon and Kim 2002). Clearly, chemically modified chitosans such as N-carboxymethyl chitosan, methyl pyrrolidinone chitosan, N-phosphono methyl chitosan, and N-carboxymethyl chitosan limit fungal growth in plants (Lafontaine and Benhamou 1996). Chitosan has the properties of enhancing the plant’s growth and reducing the diseases borne by the pathogenic soil. He noted that fungal species such as Fusarium oxysporum and Radicis-lycopersici, bottled up by the modification of chitosan, made the root rotten (Fig. 14.4). The low molecular weight chitosan acts like a highly potential biotic elicitor, recognising plants (pathogen signal metabolites) that help activate plant defences. These mechanisms try to activate multiple pathways that increase resistance to crop diseases. Falcón-Rodríguez A.B observed that, by synthesising new enzymatic molecules, the defence process produces a chemical and mechanical barrier for agricultural plants. Furthermore, the biopolymer induces the hypertensive effect of destroying the microbes around the infection region. They recently found that, accompanied by the synthesis and accumulation of secondary metabolites, the defence mechanism. Callose, phytoalexins, lignin, PR proteins, and the enzymes involved in the process are PAL, peroxidases, and chitinase, which are the main compounds used in the self-defence process. The plant cell receptors recognise the elicitors through the transmembrane cell, but the apt mechanism was not yet elucidated properly. Figure 14.2 shows the interaction between DNA and chitosan for the direct evocation on activity of gene activity in cell defence. The predictive

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model shows that by altering the structure of DNA along with the transcriptional factor of a mobile group HMG, chitosan induces the self-defence mechanism.

14.6.2 Chitin and Chitosan and Their Derivative Compounds Used for Chlorophyll Enhancement Chitosan polymeric substance extensively used in the leaves of different plants such as soya bean, coffee, pepper, black pepper, and cabbage (Bittelli et al. 2001, Wongroung et al. 2002,) The photosynthetic process began in the organelles present in the leaf called chloroplast in plant saplings. The chloroplast consists of carotenoids and chlorophyll. The critical role in the synthesis of sugars and organic compounds by fixation is played by these chloroplast compounds. Chitosan and chitosan polymer interactions increase the amount of chlorophyll and the strength of photosynthesis. Dzung (2004a, b, 2005) delineated that the 2 kDa molecular weight chitosan used in the cultivation of peanut and soya beans enhances the percentage of the chlorophyll content 18–23% and optimised concentration for the chlorophyll at 30 ppm. With the addition of chitosan around 40–50 ppm, many plants such as black pepper and coffee increases their antimicrobial activity. Table 14.6 shows the amount and effect and on concentration of chitosan oligomers present in induces the chlorophyll in some plants. Nitar et al. (2004) investigated property of the leaves of chlorophyll improved by the addition of chitosan around 20 ppm for 1 day. It is observed that the colour of the leaves is greater than control.

14.6.3 Stimulating Seed Germination It is one of the most effective methods for stimulating the germination process and is by increasing the bioactivity of chitosan and its derivatives. This phase of bioactivity was started in 1996. Hadwiger et al. (1986), Yano and Tsugita (1988) observed the chitosan derivative such as depolymerised chitin, and hydroxyethyl-chitin, CM-chitin enhanced the germination of soya bean around 6%. The efficacy of seed germination was investigated by Li and Wu (1998) using various parameters such as the degree of acetylation, the percentage of chitosan used, and the organic acid used. Different plants, such as cabbage, mung bean, radish, lettuce, green pea, alfalfa, and spoon cabbage, were involved in this research. He observed that Table 14.6 Effect of chitosan concentration to enhance the photosynthesis process Conc. of Chitooligomer Peanut Soya bean Coffee Coffee seedling

0 ppm 4.35 2.76 2.24 2.21 2.18 1.05

20 ppm 5.42 3.16 2.27 2.24 1.15

30 ppm 5.75 3.23 2.59 2.13 1.43

40 ppm 5.18 3.10 2.31 2.453 1.70

80 5.08 2.92 2.31 2.18 1.38

100 – 2.14 2.52

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germination is found in the system which was dispersed in chitosan, DD, and acid 1% acetic acids. But the researchers discovered that they were not mediated in the absence of acetic acid. Similarly, the same research was conducted to improve wheat seed germination by coating chitosan for 15 min with a molecular concentration of 2–8 mg/L. Finally, he found that the untreated wheat has reached 50–57% and the treated gains 70–90%. Sarathchandra and Jaj (2004) did the experiment to observe the germination of treat pearl millet by fixed concentration of chitosan around 4%. The millet seeds were immersed in different chitosan solution which was prepared by different concentrations such 1:5, 1:10, 1:15, 1:19, and 1:25 respectively, soak time is around 3–9 h. Eventually, the dilute sample at 1: 19, soaking time: is around 6 h shows a significant increase in seed germination around 91% compared with control is around 83%.

14.6.4 Improve Mineral Nutrient Uptake of Plants Dzung (2005) recorded that chitosan and its derivatives were squirted with a 25-ppm concentration on the cabbage leave, and then about 11% of the amount of nitrogen diffused into the leaves of the cabbage. Li and Wu (1998) defined that the components present in spoon cabbage would never be altered, even if the seed was coated with chitosan or in the case of successful yield treatment. Likewise, a similar kind of studies was carried out by Li and Wu (1998). He researched this about 20–80 ppm of the chitosan oligomer sprayed on leaves. This was delineated by the results obtained; the amount of nitrogen content increased by the accumulation of minerals in the plants about 3.3–13.6%, respectively. Chitosan oligomers have shown an effective capacity of about 50 ppm for enhancing mineral assimilation in coffee. In the coffee leaves, the addition of 50 ppm chitosan helps to accumulate about 9.49%, 11.76%, 37.7%, and 18.75%, respectively, of nitrogen, potassium, calcium, and manganese.

14.6.5 Chitosan as Soil Amendment From the previous discussion, we demonstrated that the biopolymer of chitosan has the potential to minimise microbial activity and helps to retain bioproducts without altering their characteristics and features. In addition, the mixing of chitosan on the surface of the soil often has a direct impact on reducing microbial levels and improving physiochemical conditions. Several researchers observed that, after adding the biopolymer, the growth of fungi such as Cylindrocladium floridanum, Aspergillus flavus infections, and Alternaria solani in the soil is effectively reduced. The assimilation of the soil’s chitosan biopolymer serves as an elicitor, which provides a signal to trigger the plant’s DNA self-defence mechanism. Similarly, the same kind of mechanism was observed between the enzymes, namely G-POD, PPO, PAL, and APX with the plants. Chitosan’s alteration of soil characteristics is an environmentally responsible

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process before the chitosan of the soil is deteriorated. Owing to the presence of bacterial flora present in the soil, the chitosan was blended into the soil. Several studies confirm that the existence of chitinase enzymes is primarily due to the diversity of bacterial communities in a significant fraction of the population. The bacterial and fungal species can degrade chitin molecules widely known as chitinolytic process. Chitin promotes the development of the bacteria as compared to the fungi in this process. Chitin is effectively degraded by the key bacteria, namely Bacillus species, Streptomyces, and Stenotrophomonas, but the mechanisms are not clearly described properly. The chitin changes the equilibrium conditions with the fungal species in the realistic situation and demonstrates an antimicrobial impact on some pathogens and is even more advantageous for many other bacterial species such as Bacillus, Pseudomonas fluorescens, actinomycetes, mycorrhiza, and rhizobacteria. There are two hypotheses formulated by the researchers based on the mechanism found between the chitosan and fungi (a) Chitinolytic microorganisms have the ability to break down pathogenic bacteria’s chitinous hyphae through hydrolysis. (b) Chitosan secondary products have a negative effect against pathogens (Dzung 2005; Li and Wu 1998).

14.6.6 Methods of Application of Chitin, Chitosan, and the Derivatives for Agriculture In water and even in diluted organic acids, such as lactic acids and acetic acids, the chitosan biopolymer and its derivatives typically have greater solubility. That is why chitosan is easily used for many purposes, such as soil enrichment, foliar spraying, agricultural process supplementation, seed coatings, and even material supplements used in plant culture media. It is very important to know the difficulties in the application of agriculture and standardisation of chitosan due to its growth elevations, enhancement in yield of crops, degree of deacetylation, molecular weight, and concentration of chitosan. The preferable concentration of chitosan used in coating on seed surface is about 0.2–2%, for the soil improvement 0.1–0.5%, percentage of chitosan (Mw 20–50 ppm) used in foliar spraying 0.1–0.2%, concentrations of oligomers of the chitosan (molecular weight 2–3) for spraying process, concentration of chitosan oligomer used in plant tissue culture around 15 ppm and higher Mw is preferred in high concentrations.

14.7

Conclusion

Chitosan is a biopolymer derived from chitin deacetylation found in different organisms, such as fungi, prawns, and bacteria. Chitosan can be used in various sectors, such as the food, pharmaceutical, agricultural, cosmetic and wastewater treatment industries. The ability or effectiveness of chitosan depends on the molecular weight, the solubility, the barrier of moisture, and the degree of parameters of deacetylation. Chitosan has a greater potential for antimicrobial activity and

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increases the shelf life of various products, such as bread, seafood, meat, vegetables, and pharmaceuticals. The chitosan derivatives also show a promise effect on antimicrobial activity and helps in increasing shelf life of various products. Chitosan helps communicate the self-defence mechanism for plants in the agricultural sector, increases the lifetime with coating, increases the accumulation of nitrogen sources, decreases the creation of anaerobic conditions within vegetables by reducing the surface permeability. Finally, via the enzymatic process, the chitosan can be easily degraded.

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Role of Microbial Extracellular Polymeric Substances in Soil Fertility

15

Alok Bharadwaj

Abstract

In the present scenario, conventional chemical polymers are substituted by biopolymers having various characteristics features such as high effectiveness, rapidly biodegradable, nontoxic, and possess no pollution. Presently, microorganisms used for producing extracellular polymeric substances along with biopolymers have been studied by many research workers as an effective flocculent having its application in relevance in different types of water, polluted water, and water treatment practices. These polymeric substances are extremely hydrated polymers which are secreted by a number of microbes that are chiefly made up of DNA, proteins, and polysaccharides. Tocarryout different microbial activities, extracellular polymeric substances play an important role as it provides ideal conditions for performing chemical changes, nutrient availability, and shield them from environmental stresses like drought and salinity. Moreover, it (microbial extracellular polymeric substances) increases the soil aggregation ability and assists plants by trapping moisture and nutrients. In addition to this, it also possesses some distinctive features like biocompatibility, potential to have gelling and thickening ability along with its industrial applications. Besides this, the ability of extracellular polymeric substances has been analyzed and it has been observed that only a small amount of them are being utilized in various areas, mainly in agriculture. In this chapter, an effort has been made to impart recent information associated with the various characteristics of microbial extracellular polymeric substances in the betterment of soil health by improving soil fertility and its physicochemical properties.

A. Bharadwaj (*) Department of Biotechnology, GLA University, Mathura, Uttar Pradesh, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_15

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Keywords

Biopolymer · Exopolysaccharide · Soil aggregation · Soil fertility

15.1

Introduction

Nowadays chemical flocculants have been replaced by certain microbial metabolites like extracellular polymeric substances because microbial metabolites are cheap, eco-friendly, and environment friendly. These are the biopolymers (Wingender et al. 1999). It has been clear from the previous literature that the extracellular polymeric substance matrix is produced as microbial secretions, which contains various biochemicals, discharge of different cell components as well as organic compounds present in the culture medium. In addition to it, these microorganisms, whether prokaryotic or eukaryotic, naturally secretes these extracellular polymeric substances (Flemming and Wingender 2001). Normally, an extracellular polymeric substance is found as a viscous biofilm matrix that contains 50–90% of the total organic matter. On the basis of its organization with the cell or the procedure applied for its extraction from the cell, extracellular polymeric substances classified as capsular extracellular polymeric substance (CEPS), slime extracellular polymeric substance (SEPS), loosely bound extracellular polymeric substance (LBEPS), and tightly bound extracellular polymeric substance (TBEPS) (Table 15.1). These extracellular polymeric substances are secreted by certain microbial strains act as polymers, i.e., made up of mainly DNA, proteins, and polysaccharides and it has been noticed that the secretion of these substances are elicited by the environmental signals. The biosynthesis of these extracellular polymeric substances required a high amount of energy. So, these substances must provide some benefits to the microbe that produced it (Flemming and Wingender 2010). Henceforth, the Table 15.1 Extracellular polymeric substances: types and functions S. No. 1

Type of extracellular polymeric substance Capsular extracellular polymeric substance

2

Slime extracellular polymeric substance

3

Loosely bound extracellular polymeric substance

4

Tightly bound extracellular polymeric substance

Specific function Capsular extracellular polymeric substances are an essential portion of the cell membrane and attached to the bacterial cell When biomass is centrifuged, the supernatant contains slime polymers. These are present in soluble form These are also called soluble extracellular polymeric substances that are produced in the surrounding environment in dissolved form These extracellular polymeric substances are firmly attached to the cell. e.g., sheaths, capsular polymers, condensed gel

References –

Tian (2008), Wingender et al. (1999) Tian (2008), Wingender et al. (1999) –

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manufacturing and utility of these extracellular polymeric substances are of immense importance for decades. In previous studies, some researchers have defined the functions of extracellular polymeric substances like these substances helps in attachment with any surface, the formation of bacterial aggregates, promote biofilm formation along with cell-cell recognition, proactive barrier for cells, minimizing water loss, reducing cell desiccation, and promote sorption of inorganic and organic components and ions (Tian 2008). Extracellular polymeric substances are mainly composed of proteins, carbohydrates, and humic materials. Due to the presence of such components, the extracellular polymeric substance matrix exhibit adsorption capability, biodegradability along with hydrophilicity/hydrophobicity. Moreover, extracellular polymeric substances displayed significant function in biofilm development, mass transfer through biofilm, surface deposition of various metals, organic and inorganic compounds along with providing structural support to the biofilm (Czaczyk and Myszk 2007; Flemming and Leis 2003; Neyens et al. 2004; Flemming et al. 2005). As explained earlier, one of the major functions of extracellular polymeric substances is the collection of soil particles (aggregation), which enhances soil fertility by improving the soil physicochemical characteristics. The slimy texture of extracellular polymeric substance act as a glue that helps in attachment with ions and clay that holds the solid particles together (Chenu 1995). Likewise, the structure of extracellular polymeric substances are quite variable, and henceforth their effectiveness also varies accordingly in the soil. These extracellular polymeric substances are formulated in vitro conditions for improving soil health, structure, fertility, and yield. In the present review, an effort has been made to explain the various functions of extracellular polymeric substances and their utility in the aggregation of soil particles. Many research workers have already described the ingredients, biosynthesis, and other aspects of extracellular polymeric substances in detail (Nouha et al. 2017; Flemming et al. 2016; More et al. 2014; Sheng et al. 2010; Flemming and Wingender 2010; Wingender et al. 1999b).

15.2

Ecological Characteristics

The term EPS is an acronym for “extracellular polysaccharides,” “exopolysaccharides,” or “exopolymers.” The extracellular polymeric substances are mainly produced by bacteria, cyanobacteria, algae (Boonchai et al. 2014; Parikh and Madamwar 2006), fungi (Elisashvili et al. 2009; Hwang et al. 2004), yeasts (Pavlova and Grigorova 1999), and protists (Lee Chang et al. 2014; Jain et al. 2005). Extracellular polymeric substances are basically constituted from carbohydrates, enzymes, proteins, lipids, humic acid, etc. (Flemming and Wingender 2010; Wingender et al. 1999a, b). As discussed earlier that the biosynthesis of extracellular polymeric substance is energy-based process, and henceforth the producing microbial strain must get some benefit in the environment. It has been observed in laboratory cultures that there is no

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Drought Protection

Antibiotic Protection Carbon source

Genetic Exchange

Signaling

Adhesion

Biofilm structure

Pathogenecity/ virulence factor

Aggregation Symbiosis with plants Trap of nutrients

Fig. 15.1 Microbial extracellular polymeric substances (EPS): Various advantages in soil

impact of extracellular polymeric substances on cell viability and cell growth, which means they are not necessary for existence. Moreover, it has been found that under natural conditions mostly microorganisms exist in aggregates like flocs and biofilms. Such structures are formed by extracellular polymeric substances that are structurally and functionally necessary (Wingender et al. 1999b). The most common efficiency of extracellular polymeric substances is to provide protection to the producing microorganism. The synthesis of extracellular polymeric substances can be triggered up by variation in abiotic environmental factors like pH, temperature, drought, and salinity (Vardharajula and Ali 2015; Kumar et al. 2007; Wingender et al. 1999b). The functions of extracellular polymeric substances are precise in Fig. 15.1.

15.3

Impact of Extracellular Polymeric Substances on Soil Aggregation

15.3.1 Role of Microbial Population on Soil Aggregation Soil is the uppermost layer of earth-crust and soil aggregates are the fundamental element of soil structure that is made up of pores and the solid material formed by the rearrangement of small elements, flocculation, and cementation. These are the major components that express the physicochemical properties of the soil like soil temperature, soil moisture, water holding capacity, aeration and influence the physical, chemical, and biological processes (Tang et al. 2011; Alami et al. 2000). Soil aggregates play a vital role in the enhancement of the soil porosity, productivity, and crop yield via increasing the growth of plant root and shoot (Bronick and Lal 2005; Dinel et al. 1992). The process of development of these soil aggregates depends on various factors like soil microorganisms, soil vegetation, and cation exchange between organic matter and soil particles (Kumar et al. 2013). The consistency of these aggregates is based on their porosity, internal organization, pore wall hydrophobicity, and tortuosity (Chenu and Cosentino 2011). Improved aggregation along with superior soil organization is the essential requirement of sustainable agriculture and environment (Amézketa 1999). On the basis of the hierarchical model, it can be advocated that huge (large) aggregates are constituted from the combination of smaller aggregates (Tisdall and Oades 1982). These smaller aggregates (size 2–20 mm) are constituted by the amalgamation of clay component with inorganic amorphous binding element like various oxides,

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aluminosilicates, humic materials, soil polysaccharides along with metal ions. Such long-lasting smaller aggregates combined with each other leads to the formation of larger (big) aggregates (size 20–250 mm) with the help of fungal hyphae and plant root hairs. It has been observed that these smaller aggregates stick with each other with the help of transient binding agents like polyuronides and polysaccharides to constitute large aggregates (size >250 mm diameter). There are several factors that influenced the process of aggregate formation like microbial population, organic and mineral components, plant variety, and soil type (Tisdall and Oades 1982). The major component that engaged in the formation of small aggregates are found to be clay minerals, CaCO3 and Fe- and Al-(hydr)oxides (Totsche et al. 2018). Extracellular polymeric substances are found to be associated with the development of organo-mineral complex in the soil that can affect the amount of organic substance along with the reactivity of minerals (Liu et al. 2013). The mineral aggregate of extracellular polymeric substances develop an association between various minerals and increasing their capability to hold more water by adsorbing onto the mineral surfaces (Henao and Mazeau 2009). In numerous studies, the adsorption of extracellular polymeric substances was performed and visualized through microscopy. It has been found in the previous research that electrostatic bonding is necessary between the extracellular polymeric substance of Pseudomonas putida X4 and goethite, minerals kaolinite and montmorillonite (Lin et al. 2016). At low pH, due to the protonation mechanism, the extracellular polymeric substance of C, N, and P portion showed maximum adsorption capacity. Moreover, to study the division of proteins, polysaccharides, and nucleic acid in mineral–extracellular polymeric substance complexes, the confocal laser scanning microscopy (CLSM) technique is used by the authors. Liu et al. (2013) observed that adsorption of extracellular polymeric substance from Bacillius subtilis168 to goethite and found that goethiteadsorbed extracellular polymeric substance complex becomes enriched with extracellular polymeric substance portion that mainly contains proteins and lipids but lacking in polysaccharides. The adsorbed proteins were found to be sulfur-rich that can be obtained from sulfur enriched amino acids. Whereas, the pure extracellular polymeric substances were found to be rich in protein and polysaccharides having very less quantity of lipids and nucleic acids. Research has been carried out for many years to know various microbial populations present in different aggregate categories through various experimental setup and techniques (Blaud et al. 2012, 2017). Some literature has been available on the type of microbial population that reside in aggregates of various size in different agriculture management systems (Kravchenko et al. 2014; Mummey and Stahl 2004; Sessitsch et al. 2001); on the other hand, there is no study available that describe the type of microbial population accountable for aggregate formation. It has also been reported that the properties of aggregation of soil is dependent on the isolated microbial species and such studies like impact of microbes and their extracellular polymeric substance on soil aggregation have been studied for decades. In one study, bacterial strains were isolated from two agriculture ecosystems (9 years no tillage and 40 years tillage) by using smaller aggregates (250–50 mm) and check their aggregation capability by using fatty acid

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methyl ester (FAME) technique (Caesar-TonThat et al. 2007). They found that isolated microbes like Stenotrophomonas, Sphingobacterium, Bacillus, and Pseudomonas species have the potential to stabilize and enhance the capacity of aggregate in artificial aggregates and these bacterial strains were found freely in the normal soils. Another study has revealed that there were several factors like tillage, irrigation, and cropping system that influenced the bacterial strains that reside inside the soil aggregate (Caesar-Tonthat et al. 2014). They have isolated more than 50 soil aggregating bacteria out of 1296 isolates, out of which dominating bacterial strains isolated belonged to Bacillus spp. and Pseudomonas spp. In other studies, it has also been found that Bacillus species and Pseudomonas species have the potential to produce extracellular polymeric substances and biofilms, i.e., responsible for improving soil structure and soil health.

15.3.2 Inoculation of Extracellular Polymeric Substance Producers in Soils From previous research, it has been proved that in the process of soil aggregation and stabilization, microorganisms play a very important role. Moreover, there are many factors like type of microbial species, available soil nutrients, and their management that influence the microorganisms that are responsible for soil structure management and stabilization (Beare et al. 1994; Umer and Rajab 2012). Among the microbial community, mainly bacteria and fungi secrete extracellular polymeric substances and carry out breakdown of aromatic humic substances that produces clay–metal– organic matter complexes which are ultimately required for the stabilization of soil structure (Umer and Rajab 2012). In addition, fungi contribute with the help of their hyphae although with less perseverance. In previous studies, a number of bacterial and fungal species have been utilized for representing the impact of pure microbial cultures on soil aggregation and it has been found that the whole process is dependent on the type of microbial strain. Henceforth, for improving the soil productivity and soil quality, various microbial strains and extracellular polymeric substances have to be searched out for the components that increase the aggregation capability in various types of soils. From previous research, it has become evident that the bacterial strains, that produce extracellular polymeric substances and have the capability to produce soil aggregates belong to the genus Bacillus, Pseudomonas, and Paenibacillus. These bacterial strains can be easily grown at in vitro conditions and secrete a large quantity of extracellular polymeric substances. Moreover, some fungal species like Penicillium and Streptomyces also exert a noteworthy positive impact on erodibility and soil loss after rainfall simulation (Gasperi-Mago and Troeh 1979). It has been found that inoculation of Pseudomonas putida strain GAP-P45 in soil enhances the soil aggregation capacity and its stability in more than 50% cases and it depends on certain factors like salt, temperature, and drought stress. Moreover, during the situation of stress microbial strains secretes large amounts of extracellular polymeric

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substances that help the bacteria in defending it from water stress and improved soil structure (Vardharajula and Ali 2015). Another study was performed from the Gurbantünggüt Desert, where an unknown bacterial strain has been isolated from biological soil crusts (BSCs). This bacterial strain not only stabilizes the sand surface but also increases the aggregation process along with decreasing the soil water evaporation only after 8 days of inoculation. Moreover, the extracellular polymeric substances secreted by the bacterium possess a glue-like substance that helps in binding the soil and sand particles that can be examined by scanning electron microscopy (HuiXia et al. 2007). Another study was performed on Paenibacillus KLBB0001 that secretes a large quantity of extracellular polymeric substances at Gurbantünggüt Desert. In this study this bacterial strain when inoculated in desert soil, it improves the recovery of biological soil crusts (BSC). It has been observed after 1 year of experimentation that soil becomes enriched with a higher number of bacteria, phosphorus, and available nitrogen. Moreover, at the inoculation area, some sticky substance aggregating sand and soil particles showed the presence of extracellular polymeric substance confirmed from the microscopic studies (Wu et al. 2014). Several studies revealed that in place of pure culture, a mixture of microorganisms may be an alternative approach for the same. Swaby (1949) found in his study that when mixed microbial culture was applied in soil, the rate of aggregation of soil particles maximized may be due to interaction between various microbial strains. Different microbial species have different types of extracellular polymeric substances produced. When the extracellular polymeric substance of one microbial species come in contact with the extracellular polymeric substance of other microbial species like extracellular polymeric substance-coated fungal hyphae, the resulting product has a greater aggregation capability of binding the soil and sand particles together in comparison to physical involvement by hyphae (Aspiras et al. 1971). In addition, the amalgamation of microbial inoculants with organic fertilizers enhances the microbial aggregation capability, resulting in increased production of extracellular polymeric substances, ultimately ensuring improved soil fertility, structure, and function (Rashid et al. 2016).

15.3.3 Inoculation of Extracellular Polymeric Substance Producers in Plants Another important and latest available technology for sustainable agriculture is the inoculation of plant with arbuscular mycorrhizal fungi and plant growth promoter rhizobacteria. In this technology, microbial strains established themselves within plant roots increase their population along with the secretion of exudates. The population of microbial species enhanced in the rhizosphere region, as a result of the liberation of organic carbon by the plant roots. In response to it, a gelatinous extracellular polymeric substance is produced that encourage soil aggregation capacity and increasing root-adhering soil (RAS). An environment has been established by the RAS aggregation, through which plants utilize water and nutrients for their

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growth and expansion. The availability of these advantageous microorganisms helps the plants in the accessibility of major nutrients like N, P, K, and iron for their growth and expansion (Rashid et al. 2016). The data available on the recent research clearly revealed that the most promising bacterial strains, that secretes large quantity of extracellular polymeric substances and increase plant growth belong to the genus Rhizobium, Pseudomonas, Bacillus, and Pantoea. These bacterial strains when added either directly into the soil or in seedling exerts the optimistic approach on the plant development and growth also (Cipriano et al. 2016). The extracellular polymeric substances secreted in the rhizospheric region protect the plant from desiccation, variation in the water potential, rising nutrient uptake as well as supports plant growth. With the enhancement in the soil aggregation capability, the soil structure improves that ultimately enhances the growth and yield of seedlings because it encourages the uptake of water and nutrients (Sandhya et al. 2009; Bezzate et al. 2000; Alami et al. 2000). A lot of research has been carried out on PGPR and AMF but at that time the major emphasis was on plant growth but not on soil aggregation. Rhizobium strain KYGT207, i.e., a wheat growth promoter and extracellular polymeric substance secreting bacterium was picked from arid soil in Algeria. This bacterium is capable of improving the soil structure and its productivity. With the inoculation of Rhizobium strain KYGT207 strain in wheat plant, the rootadhering soil dry mass/root dry mass ratio was increased by 137%. In addition, it also increased the percentage of water stable aggregates by reducing the soil water stress through the secretion of extracellular polymeric substances (Kaci et al. 2005). These inoculated microbial species interact with the microorganisms that are naturally present in the rhizosphere region and showed a considerable impact on soil characteristics and quality by enhancing plant productivity. Unwanted germination and root growth must be inhibited for getting improved soil structure and increased aggregate formation. The use of these microbial inoculants has been continued for the last several years but more efforts must be required for providing optimum conditions for the growth of a particular microorganism to obtain more efficient inoculants, better biomass, and quality extracellular polymeric substances. Henceforth, these strains gave a maximum performance in soil with an increase in their population and become dominant on the natural population of microorganisms.

15.3.4 Inoculation of Pure Extracellular Polymeric Substance into Soil Previous research also connects the products of microbial secretion with soil aggregation capability. For improving the soil structure and productivity, polysaccharides also play an important role, although they are not the major soil aggregating molecules. Moreover, humic acid has the ability to enhance the soil aggregation capacity as well as improving soil structure. When natural and synthetic soil aggregates were treated with certain chemicals like tetraborate and periodate, they do not show a stable pattern as polysaccharides are necessary components required

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for a constant soil aggregation process (Sparling and Cheshire 1985; Mehta et al. 1960). To prove this statement, an experiment was performed for observing the correlation between carbohydrate content and mean weight of soil aggregate, and the results obtained were clearly depicted that for enhancing the capability of soil aggregation, carbohydrate content was essentially required in soils with various crops (Angers and Mehuys 1989). Moreover, the addition of sodium periodates in soil before wet sieving also mentioned the contribution of carbohydrates in maintaining the stable soil aggregates. Biopolymers, that are resistant to breakdown mechanism may contribute to the improved soil structure. In other words, we can say that the greater is the resistance, the extended is the longevity of extracellular polymeric substances in soils. Colloids and metal ions present in the soil may affect the degradation of biopolymers. Now investigation was initiated for the inoculation of polymers in the soil, it has been found that there is variation in the binding capacity of microbial polysaccharides and plant itself. Nevertheless, certain parameters of the soil like pH also affect the mechanism of polysaccharides’ action. It has occurred due to the charge on the molecules that is mandatory for binding of the particles (Martin 1971). There are certain features of polysaccharides that affect the binding capacity like flexible nature, linear structure, and length, that ultimately permit the Van der Waals forces, hydrogen bonding, and acyl groups, allowing the ionic binding to clays (Martin 1971). There are many bacteria and fungi that produced a variety of extracellular polymeric substances and their performance has been checked as soil aggregating agents. For this, in place of the addition of microbial strains, an alternative approach has been used by directly applying the polymers to the soil. The extracellular polymeric substances secreted by L. mesenteroides, B. subtilis, and Leuconostoc dextranicum have been evaluated for their aggregating potential by Geoghegan and Brian (1948). It has been found that various extracellular polymeric substances produced have a significant role in soil aggregation capability and were examined by wet sieving process and small quantity of levans (0.125–0.05%), that have a significant role in stabilizing the aggregates. Chromobacterium violaceum is a Gram-negative coccobacilli that secrete a specific extracellular polymeric substance which exerts more resistance to degradation than other plant polysaccharides. The extracellular polymeric substance secreted by this coccobacilli displayed the highest aggregation capacity among all polysaccharides and maintains the soil structure along with its pH (Martin and Richards 1963). There are certain extracellular polymeric substances that exert very high water holding capacity. Chenu and Roberson (1996) have demonstrated the water holding capacity of xanthan and found that water holding capacity of 15 times its weight. On the other hand, when dextran was analyzed for the same activity exerts less water holding capacity may be due to variation in structure. It has been observed from the previous research that the extracellular polymeric substances that possess higher water holding capacity can able to protect plants, soil, and microorganisms from

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drought stress, encouraging hydrating conditions and bridging between clay and soil particles. It has also been observed from the previous research that in place of direct inoculation of extracellular polymeric substances in soil, its production can be improved by N management. The impact of the incorporation of N in extracellular polymeric substances and soil aggregation has been observed by Roberson et al. (1995) by using indirect measurements and monosaccharide composition. He depicted that when intermediate and high amount of N fertilization provide the same crop yield, the data obtained for soil characteristics showed variation in the results. That is because the intermediate N fertilization encouraged improved aggregation and saturated hydraulic conductivity. Henceforth, the production of extracellular polymeric substances could be enhanced by inoculation of nutrients directly into soil ultimately improving the soil structure and soil aggregation capacity.

15.4

Conclusion

For survival in soil, microorganisms undergo various mechanisms to tolerate certain environmental stresses. Extracellular polymeric substances provide favorable conditions for the growth of microorganisms like wet atmosphere, ease of trapping nutrients, assisting chemical reactions, and provide a shield against environmental stress and predators. These extracellular polymeric substances when released by the microorganism contain a variety of compounds and perform several functions based on their organization and composition. A lot of previous research has been demonstrated that due to the production of extracellular polymeric substances, the soil aggregation capacity, soil structure, and soil quality along with soil fertility improved drastically. Furthermore, besides improving soil structure, these extracellular polymeric substances also improve the uptake of nutrients and availability of water to both microbes and plants, and henceforth not only adventitious for the plant but also the environment as a whole. As we all know that microorganisms possess huge capabilities and can be increased by improving the structure and composition of extracellular polymeric substances along with using a consortium of microorganisms for huge quantity production of extracellular polymeric substances. Due to the valuable characteristics like biocompatibility, biodegradability, emulsion, gelling, and aggregating capabilities, these extracellular polymeric substances are the choice of interest since years back. For obtaining a higher yield, manipulation was carried out in the polymers, i.e., based on description and knowledge of physiological characterization of extracellular polymeric substance producing microorganisms. A number of microorganisms produced extracellular polymeric substances but because of the change in the polymer composition, many prospects remain open for investigation and experimentation.

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Microbes Derived Exopolysaccharides Play Role in Salt Stress Alleviation in Plants

16

Purnima Singh, Vibha Pandey, and Prerana Parihar

Abstract

Several factors are responsible for the annihilation of agriculture production. Contribution of salt stress as an abiotic factor is gaining a striking position among all. The reason assigned for it is pressure on land for higher production to fetch the ever-increasing gigantic world population coupled with everchanging climatic conditions. Erratic rainfall and higher temperature in arid and steppe climatic regions are further aggravating this problem. It is estimated that more than 50% of all arable land will suffer from salinization till 2050 and every year 1.5 million ha irrigated land is falling out of agricultural production. Salinity affects plant growth by disrupting cell ionic balance that ultimately causes deviation in cell division, photosynthesis, transpiration, plant signaling system, etc. Coping with salinity is a global issue to ensure sustainable crop production. Since the major upshots of soil salinization are loss of biological activity and deterioration of physiochemical fabric. Coevolution of plant and microbes always provide an opportunity to get a sustainable answer from rich microbiota of plant rhizosphere. Halophilic and halotolerant microbes residing in salt-stressed soil are capable of improving soil health and crop productivity through secretion of several polysaccharides, viz., intracellular storage polysaccharides, extracellular polysaccharides (also called Exopolysaccharide, EPS), and capsular polysaccharides. These polysaccharides contribute to biofilm formation and pathogenicity. Artificial tailoring of polymers having unique attributes such as

P. Singh · P. Parihar Department of Plant Pathology, College of Agriculture, Rajmata Vijayaraje Scindia KrishiVishwa Vidyalaya, Gwalior, Madhya Pradesh, India V. Pandey (*) Department of Plant Pathology, College of Agriculture, Jawaharlal Nehru Krishi Vishwa Vidhaylaya, Jabalpur, Madhya Pradesh, India # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_16

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biocompatibility, gelling, and thickening as of ESP can be made superior through industrial application. Hence, comprehensive knowledge about EPS biosynthesis mechanism and its mode of action will further expand its role in salinity amelioration to address salinity problems. Therefore, microbial-derived EPS widens the scope of its use as a potential sustainable alternative for enhancement and protection of agricultural crops in salt stress arena. Keywords

EPS · Biofilm · Salt stress · Biosynthesis of EPS · Salinity amelioration

16.1

Introduction

Perpetual agriculture is indispensable for today’s world as it provides the possibility to fulfill our farming exigency. In developing nations, crop production is chiefly curtailed by the bleaker ramification of biotic and abiotic stress. Plants are sessile, so they are different kinds of biotic and abiotic stresses such as flood, dry spell, salt stress, heat and cold, exposure to heavy metals, nutrient deficiency as well as plant pathogens, and pests. Among various environmental factors, the presence of salt in the soil is one of the main issues responsible for lowering plant growth and crop productivity across the globe. As per salt-affected soil portal of FAO, the land affected by salinity throughout the world accounts for more than 1000 million hectares. The enhanced stress (high conc. of NaCl) not only led to high ethylene production but also induced ion toxicity, oxidative stress, which disturbed the osmotic potential in plants. Soil stress affects all the physiological functions like photosynthesis, nitrogen fixation, respiration, etc., which are essential for plants, resulting in a decrease in farm production and productivity. Due to the irrational application of agrochemicals, the trouble of soil salinity is getting more pronounced in arid and steppe climate. The use of PGPR is an assured biological approach to extenuate the salt stress. They can cope up with salt and other abiotic stresses by using a mechanism like induced systemic resistance, which helps them to thrive easily in harsh conditions. However, fungal diversification increases as the salt imposed stress increases upto 24.1 g NaCl/L concentration, but it was remarkably reduced to 44.1 g NaCl/L. However, club fungi (Basidiomycota) progressively replaced by Ascomycota as salinity increased while club fungi community flourished in the lack of NaCl (Cortés-Lorenzo et al. 2016). In the semiarid areas, the main restricting feature to crop productivity is the use of water which has high soluble salt content. So, the identification, exploitation, and use of halotolerant microorganisms may lower salt stress and will provide a new eco-friendly substitute for stress tolerance in crops. In the mesophilic prokaryotic Escherichia coli bacteria first halotolerant gene was described. This gene proB-74 was responsible for determining the deposition of osmolytes like proline in (Serrano and Gaxiola 1994). Similarly, Khan and coworkers (2009) have proposed long-term plan for revamping the diversity of mangroves through mechanistic application of

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EPS-producing halotolerant microbes for mitigating derelict soil and enhancing yield. EPS (exopolysaccharides) is known to produce by many algae, yeast, bacteria, and fungus. The production of EPS by many microorganisms is an immediate reaction to selective pressure exerted by the natural environment. Microbial EPS are biodegradable in nature; hence, causes no damage to the environment in comparison to synthetic polymers. This makes them environment friendly. The use of microbial polymer has begun in 1960 and thereafter there has been an exceptional enhancement in their remunerative use. Microbial EPS play a key role in micro and macro environment as well as, in cellular associations and in nutrition. EPS-producing microbes can live below the required threshold nutrient amount and better survive under an oligotrophic environment. This chapter aims to present overall information on microbial EPSs, their biosynthesis, and how they can be used to combat salt stress.

16.2

Salinity and Crop Production

Most grown crops are vulnerable to salinity stress. The term salinity remits soluble salt concentration present in soil and water. According to Mahmuduzzaman et al. (2014), in the coastal region, salt stress is a key problem decreasing the production of standing crops. Based on origin, salinization is of three types according to Zhou et al. (2013): (a) Primary salinization: (b) Secondary salinization: (c) Tertiary salinization:

natural salinity main cause is the rise in groundwater level this happens due to irrigation

Based on adaptive evolution, plants are roughly categorized into two major types: plants which can withstand salinity stress known as halophytes and second plants which cannot withstand salinity stress and die are known as glycophytes. Unfortunately, most of the agricultural crops belong to the second category. Planting soil tolerant crop like Barley and Canola is a usual practice (Fita et al. 2015). However, these crops are capable of tolerating normal salt stress, so they have restricted reach and cannot be used in region which has moderate or high soil electrical conductivity (Morton et al. 2019). According to Mass and Hoffman model the crop yield is not affected until the salinity threshold surpasses up to a certain limit. Since optimum amount of salt concentration is required by plant for growth. High amounts of salt (Na+ and Cl
) are toxic to plants. Moreover, salt concentration beyond tolerable limit (Gupta and Huang 2014) lead to crop failure as plant fails to absorb water and wilting occurs. Salt stress also influences the plant growth by disrupting cell ionic balance that ultimately causes deviation in cell division, photosynthesis, transpiration, plant signaling system, etc. Thus, salt stress is a major vicious abiotic stress that hamper crop yield around the world (Flowers 2004).

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As reflected by the growth responses of plants, they greatly show differences in their salinity tolerance. For example, cereals rice (O. sativa), foxtail barley (Hordeaum vulgare), bread wheat (Triticum aestivum), and durum wheat (T. turgidum ssp. durum) are most sensitive, most tolerant, moderately, and less tolerant to salt stress, respectively (Munns and Tester 2008). There is a greater variation in salinity tolerance in dicot plants in comparison to monocot plants. Some legumes are even more sensitive than rice (Lauchli 1984); Medicago sativa (Alfa-alfa or Lucern) is very tolerant and continues to grow at higher salinity.

16.3

EPS and Biofilm Producing Microorganism

Salt stress is critical abiotic stress which negatively influences the biochemical and physiological mechanism of plants and severely hampers crop productivity worldwide (Per et al. 2017). There are several groups of microorganism (Table 16.1) like bacteria, microalgae, cyanobacteria, and fungi that can live even in grating conditions. Production of EPPs and biofilm by halotolerant or halophilic microorganism are some added mechanisms which provide salinity resistance (Steele et al. 2014) in microbes. EPSs are a natural class of biopolymer which have great ecological importance. EPS are a general attribute of biofilm, where they have structural and protective functions (Rossi and De Philippis 2015). Microbial EPS represents a sustainable and alternative for the protection and enhancement of crops from salinity stress. The EPS has capability to attenuate the repercussions of salinity stress on the growth of agricultural crops grown in different salinity levels. EPSs alleviate salt stress by decreasing the amount of available Na+ absorption in plant. A crop when employed with EPSs exhibit enhanced soil structure and improved resistance to both salinity and water stress (Sandhya et al. 2009). Acidic EPSs have the capacity to cohere with Na+ ions and making them unobtainable to plants under stress condition (Shrivastava and Kumar 2015), these bacteria mostly produce succinoglycan-type acidic EPSs. A study on root-zone inhabiting bacteria Aeromonas hydrophila, B. insolitus, and Bacillus sp. isolated from wheat seedling growing under usara soils revealed that they considerably increased dry matter of roots and shoots. Due to enhanced assimilation of calcium, sodium, and potassium ions from salt, microbial EPSs resulted in increased crop productivity (Ashraf et al. 2004). Salt tolerant PGPR are key microbes in extenuating salt stress by inducing physical and chemical modifications. Indirectly, under stress conditions they regulate the conc. of ethylene in plants through the action of ACC deaminase (Glick 2005) and they produce siderophores to fight against disease-causing entities as well as accelerate nutrient absorption through the formation of various phytohormones (Goswami et al. 2016). A conglomerate of bacteria does so by producing bacterial EPSs which are involved in the adherence of bacteria in environmental surfaces and this association is known as biofilm (Mah and O'Toole 2001). To recoup the stress foisted by salt, salt-tolerant bacteria aid to metabolism and form biofilm and produce EPS. Halophilic bacteria can form biofilm and produce EPS at higher salt stress. EPS

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Table 16.1 EPS-producing microorganism S. no. 1.

Microbes Azotobacter chroococcum

Crop Vicia faba

3.

Dunaliella salina (microalgae) Halomonas sp.

Solanum lycopersicum Oryza sativa

4.

Halomonas variabilis

Cicer arietinum

5.

Planococcus rifietoensis

Cicer arietinum

6.

Dietzia natronolimnaea STR1

Triticum aestivum L.

7.

Bacillus alcalophilus, B. thuringiensis, and Gracilibacillus saliphilus

A. macrostachyum

8.

M. yunnanensis, P. rifietoensis, and V. paradoxus S. brachiata

Beta vulgaris L.

Pseudomonas putida (GAP-P45) Pseudomonas extremorientalis (TSAU20) and Pseudomonas chlororaphis (TSAU13) P. alcaligenes (PsA15), B. polymyxa (BcP26), and M. phlei (MbP18) Aneurinibacillus aneurinilyticus and Paenibacillus sp.

Helianthus annus

2.

9.

10. 11.

12.

13.

Remark Decreased Na+ and Cl
conc. significantly in salt stressed Faba beans 3 and 6 g L
1 NaCl

Reference Abd El-Ghany and Attia (2020)

Under both salt and arsenic stress enhanced growth Tendency to form biofilm at higher salinity Tendency to form biofilm at higher salinity Enhanced wheat tolerance to salinity stress Mitigated the effects of high salt Physiological performance of the plant Enhanced salinity tolerance

Mukherjee et al. (2019)

SbASR-1 gene transcript showed upregulation under salt stress conditions Release EPS and alleviates salt stress Reduced oxidative stress

Jha et al. (2012)

Sandhya et al. (2009) Egamberdieva et al. (2017)

–

Tolerate high temperatures and salt conc. in aridisol

Shrivastava and Kumar (2015)

Phaseolus vulgaris

Decreased stress stimulated ethylene production

Gupta and Pandey (2019)

Transgenic Tobacco

Solanum lycopersicum L.

Qurashi and Sabri (2012) Qurashi and Sabri (2012) Bharti et al. (2016) Navarro-Torre et al. (2017)

Zhou et al. (2017)

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encourages bacterial colonization near plants root, and they can finally be added in soil. Till now, the rhizobial EPS synthesis has been studied in two specie S. meliloti and R. leguminosarum (Janczarek 2011). It is well documented that nutrient and other stress conditions are responsible for biofilm formation in S. meliloti and R. Leguminosarum. In recent decades, many microalgal extracellular polysaccharides have been reported and their functional properties were documented. A number of microalgae like Arthrospira sp. (Spirulina), Botryococcus braunii, Chlamydomonas reinhardtii, Dunaliella salina, Dunaliella tertiolecta, Isochrysis galbana, and Porphyridium cruentum can be a fount of EPSs (Rossi and De Philippis 2016). Ethylene is produced in multiple stresses and is also associated with the management of many physiological activities of crops, but the ethylene production in plants is influenced by climate change, which imposed a notable depletion in crop growth and development (Iqbal et al. 2017; Dubois et al. 2018). ACC is the precursor of ethylene synthesis, which is secreted as root exudates by plants, growing under stressful conditions (Wang et al. 2013; Liu et al. 2015; Abiri et al. 2017). ACC deaminase has the capacity to enhance plant growth and development which are surviving under adverse conditions (Gupta and Pandey 2019). The presence of more amount of salt in soil stimulates the biosynthesis of ethylene by precursor ACC; hence, it is called salt stress-induced ethylene (Chookietwattana and Maneewan 2012). PGPR bacteria can lower salt stress by reducing the concentration of ethylene. Ammonia and α-ketobutyrate are formed after the breakdown of ACC by an enzyme ACC deaminase that is present in certain PGPR; therefore, lowering the conc. of ethylene within the plant. Therefore, they have the capability to suppress the abiotic stress induced by ethylene production and its associated harmful effect on plants. There are many research papers suggesting that plants when inoculated with PGPR having ACC deaminase make the plant more resistant to different abiotic stresses. Cyanobacteria are prokaryotic, unicellular or filamentous, oxygen-producing microorganisms among which some of them can fix atmospheric nitrogen. Green algae are eukaryotic lower plants. Numerous cyanobacteria and green algae are surrounded by a special mucilaginous covering around their cells or filaments. Filaments are mainly made up of carbohydrates which are synthesized and secreted extracellularly. Many studies have analyzed the composition of EPS using various chromatographic and mass spectrometric techniques. The principal sugars present in EPS of cyanobacteria and microalgae are glucose, galacturonic acid, fucose, galactose, rhamnose, arabinose, xylose, mannose, orthomethyl sugar, and acidic residues of glucuronic acid. Depending upon species EPS form different types of layers around the cells and called as slime, sheath, or capsules (Drews and Weckesser 1982; De Philippis and Vincenzini 2003). In fields of science and industry, for fungal PSs there are many research avenues open (Osin’ska-Jaroszuk et al. 2015). The information on the diversity of fungal EPS, their mode of action and mode of interaction with higher organisms are still finite; therefore, there is a need to explore the efficacy of the EPSs produced fungi.

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Chemical Structure of EPS

A wide range of bacteria secrete EPS which are carbohydrate polymers, which have an important role in symbiosis and pathogenesis. The EPSs can be homopolymer or heteropolymer and may have a diversity of non-carbohydrate substituents. The most common homopolymers found in plant interactive rhizobium bacteria family are the periplasmic {3-1,2 glucans (Hisamatsu et al. 1987), cellulose in Agrobacterium (Matthysse 1983) and Rhizobium species, levan in Erwinia (Bennett and Billing 1980) and Pseudomonas (Fett et al. 1989) species, and some alginates in fluorescent Pseudomonas sp. (Fett et al. 1989). Based on location in bacterial cell EPSs may be assembled into two groups: extracellular or intracellular. The intracellular biopolymers are less and have limited utilization in comparison to extracellular biopolymers. The extracellular biopolymers can be grouped into four major classes; (i) polyamides (ii) polyesters (iii) inorganic polyanhydrides, and (iv) polysaccharides (Cerning 1995; Rehm 2010) and are commonly known as extracellular polymerhic substances (Rehm 2010), slime, and microcapsular polysaccharides (Sutherland 1972; Ruas-Madiedo et al. 2002) by others. Among the four components of the extracellular biopolymer, polysaccharide component is the most abundant that forms the basis of its classification. Their presence on the cell wall serve as protective and structural functions and is found as one of the constituents of teichoic acid. Outside the cell, they may take a shape of the morphological entity known as capsule (Ruas-Madiedo et al. 2002) or completely release in the environment as a slime layer (Cerning 1995). Fungal Polysaccharides are classified based on their systemic propinquity, structure, sugar composition (homo and heteropolysaccharides), bonds present [β(1-3), β(1-6) and α-(1-3)], and their location in cell (cell wall polysaccharides, exopolysaccharides, and endopolysaccharides). Exopolysaccharides (EPSs) are the most studied fungal polysaccharides but their definition, origin, and classification are unclear and need more explanation (Osinka-Jaroszuk et al. 2015). The different ecological functions are performed by Ascomycota and Basidiomycota. Therefore, EPSs play a vital role in different biological functions with other organisms and host plants. The biochemical and biological properties of synthesized EPSs by Basidiomycota and Ascomycota manifest a lofty of variation (Donot et al. 2012; Mahapatra and Banerjee 2013).

16.5

Biosynthesis of EPS

EPSs are synthesized intracellularly throughout the growth phase. Microbial EPSs are generally synthesized in four (Stanford 1979) steps, involving four different types of enzymes (Kumar et al. 2007); 1. 2. 3. 4.

Uptake of a specific substrate (e.g., glucose). Metabolism of sugar proceeds and the substrate is phosphorylated. Polymerization. Modification and release.

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The rate of EPS production depends upon the stress and microbes produce different types of EPS by using different biosynthetic pathways. The genes which organize biosynthesis of EPS are generally clustered inside the genome of the particular producing organism. Based on biological functions polysaccharides produced by microbes can be classified into capsular polysaccharides, intracellular storage polysaccharides (glycogen), extracellular bacterial polysaccharides which are also called as exopolysaccharides (alginate, cellulose, xanthan, sphingan, etc.) which are necessary for biofilm formation and pathogenicity. Mannose, galactose, and glucose are some common monomers found in polysaccharides of various species. Amino sugars, neutral sugar (galacturonic), uronic (fucose and rhamnose), ester-linked substituents, and pyruvate ketals are EPS components (Abbas and Naqqash 2019). Till now four general mechanisms are known for the synthesis of carbohydrate polymers: (i) ATP-binding cassette (ABC) transporter-dependent pathway (ii) Wzx/Wzy-dependent pathway (iii) extracellular synthesis by use of a single sucrase protein, and (iv) synthase-dependent pathway. Since it is known that acidic EPS binds to ions present in saline soil, this chapter will only focus on the biosynthesis of acidic EPS. The acidic heteropolysaccharides produced by rhizobia has a complex structure as well as the synthesis process. Wzx/ Wzy-dependent pathway produces acidic EPS succinoglycan in several bacterial strains like Pseudomonas, Alcaligens, Agrobacterium, and Rhizobium (Harada and Yoshimura 1964; Zevenhuizen 1997). For the production of succinoglycan, the model organism discussed in this chapter is R. meliloti. Succinoglycan (Fig. 16.1) is a branched heteropolysaccharide consists of repeating units of an oligosaccharide and acetate, succinate and pyruvate are present as many alternative decorations. The monomers present in the repeating unit are β-linked glucose and galactose and they are present in the 7:1 ratio. According to Stredansky (2005) pyruvate present in a stoichiometric ratio, while depending up on cultural conditions and strain, the presence of acetate and succinate decoration depends. Succinoglycan is essential in plants as it is necessary for plant symbiosis and other biological functions (Leigh and Walker 1994). The biosynthesis of these acidic EPS involves four major steps (Boulnois and Jann 1989; Jann and Jaon 1991; Long et al. 1988) which are as under: 1. 2. 3. 4.

Nucleotide sugar diphosphate intermediates synthesis. Assembly of the repeating oligosaccharide subunit stepwise. Addition of decorations. Transfer of the formed polysaccharide chain from its carrier lipid to the new subunit.

Very less is known about the polymerization step, but it is assumed that polymerization occurs in cytoplasmic membrane inner face, after which Bayer adhesion zones present between the inner and outer membrane transports the EPS through it. A total of 19 sets of genes are required in R. meliloti SU47 for the biosynthesis of succinogylcan, two clusters of genes are recognized, one (Long et al. 1988; Reuber et al. 1991) for succinoglycan synthesis and second (Glazebrook and Walker 1989)

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Fig. 16.1 Chemical structure of Succinoglycan

Thi P

R

H

R

N M A L

R

KH

R Bg

R

T

X

YF

R

C

HH

QZ

B

R R R

R

5 kb

Fig. 16.2 Genetic map of the R. meliloti SU47 exo gene cluster (The map is derived from Long et al. (1988)). Yellow boxes show the minimum boundaries of the exo loci designated above the line; Thi- thiamine biosynthetic locus. Vertical line -cutting sites by restriction enzymes designated below the line. Abbreviations are: R-EcoRI; H-HindIII; Bg-BglII; and C-CiaI. Not all of the sites for the latter two enzymes have been determined. Horizontal arrows indicate the direction of transcription)

for EPSb (EPS II) synthesis are found on the indigenous megaplasmid pRmeSU47b (pSymb) (Hynes et al. 1986; Finan et al. 1986). The succinoglycan synthesis genes are called as exo genes, while EPSb synthesis genes are called as exp or muc. The exo loci are necessary for the biosynthesis of acidic EPS in R. Meliloti (Reuber et al. 1991). The exo cluster has been distinguished by genetic complementation and sequencing and contains at least 12 genetic complementation groups within a 22 kb region (Fig. 16.2). Also, several loci located outside the exo gene clusters affect the biosynthesis of succinoglycan. While exp clusters contain seven complementation groups (Glazebrook and Walker 1989). Nucleotide sugar precursors are first synthesized in the biosynthesis of succinoglycan. Gene exoC codes for an enzyme phosphoglucomutase which is responsible for transferring phosphate group from the 60 to the 10 position in reverse direction (glucose-6-phosphate ! glucose-1-phosphate) (Uttaro et al. 1990). UDP-glucose-4-epimerase is encoded by gene exoB, which converts UDP-glucose to UDP-galactose (Buendia et al. 1991). Mutation in exoB and exoC causes the lack of EPS I production but also affects the synthesis of other polymers like EPS II, LPS, and β-glucans. Protein which displays UDP-glucose pyrophosphorylase activity is encoded by exoN gene. Mutation in exoN gene leads to a reduction in EPS I production (Glucksmann et al. 1993; Becker et al. 1993).

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EPS synthesis in rhizobium is regulated transcriptional and posttranslational levels. The finest distinguished transcriptional regulator acts negatively. Many researchers concluded that R. melitoti (Doherty et al. 1988; Reed et al. 1991a) has a negative regulatory locus, exoR which affects succinoglycan synthesis. On pSymb, the exoR gene is chromosomal and unlinked to the exo cluster. The existence of several independent insertion mutations leads to the negative regulatory nature of exoR gene, which causes enhanced EPS synthesis and they are recessive natured mutations. Alkaline phosphatase activities of exo-phoA fusions indicate that exoR regulates the transcription or translation of most of the genes of the exo cluster with the exception of the galE equivalent exoB (Doherty et al. 1988; Reuber et al. 1991). Another succinoglycan regulatory gene, exoS which resembles like exoR. This gene (exoS) also lack genetic linkage to the exo cluster, mutation of both the genes exoR and exoS affected the synthesis of succinoglycan and also on the expression of exo gene (Reuber et al. 1991). Genetic regulation of EPSb synthesis in R .melitoti is relatively less known. Two chromosomal mutations, mucR (Zhan et al. 1989) [probably similar to rexA (Pühler et al. 1991)] and expR cause increased EPSb synthesis. Many Rhizobium sp. has a new regulatory mechanism which is conserved and imparted by the exoX and exoY genes. The exoX (psiA)-exoY (pssA) are posttranslational regulatory system of Rhizobium sp. the exoX gene is found in R. meliloti (Reed et al. 1991b; Zhan and Leigh 1990) that encodes a small protein with hydrophilic as well as hydrophobic domains. In R. meliloti, exoX affects succinoglycan synthesis but not EPS synthesis (Reed et al. 1991b; Zhan et al. 1991).

16.6

Inoculation of EPS Producers in Soil and Plant

16.6.1 Inoculation in Soil Microbial polysaccharides are the most active organic constituents in soil aggregate formation and stabilization. According to several studies, microbes are elementary for stabilization and aggregation of soil, the effect of microbes on soil structure and stability depends upon the type of microbial species present, soil management and available substrate (Umer and Rajab 2012). The microbial polysaccharide binds the soil particle through polymer bridges. Pseudomonas, Bacillus, and Paenibacillus are the most explored EPS-producing bacterial species for soil aggregation, these genera are easily cultured in laboratory conditions and produce a decent quantity of EPS. When GAP-P 45 strain of P. putida is inoculated under stress conditions, the strain produced an increased amount of EPS in the soil as a result, it enhanced aggregate stability by more than 50% in soil (Vardharajula and Ali 2015). Under salt stress condition many cyanobateria survives by accumulating increased amount of saccharides in cells as well as outside the cell (Padhi et al. 1997). Soil aggregation rose at higher salinity up to 100 mM in inoculated soil (Qurashi and Sabri 2012) and aggregate formation was more pronounced near roots.

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This indicates biofilm formation near roots in response to salt stress leads to bacterial attachment and soil particles adhere together with roots in response to increased biofilm formation at higher EPS (Ashraf et al. 2005). This binding property of EPS would act like cementing material and provide strength to soil aggregate formation (Ashraf and Hafeez 2004; Ashraf and Foolad 2007) and favor development of plants under harsh conditions. In addition, inoculation of cyanobacteria in soil enhances the structure of the soil. Due to EPS production, these bacteria stabilize the soil surface. Moreover, the inoculation of cyanobacteria is extended up to subcrust, improves soil fertility, structure, and nutrient bioavailability. Rossi and De Philippis (2015) explored that when degraded and desertified land was treated with cyanobaceria; it improved the quality of arable land. They are the best candidate, as they possess the benefits like oligotrophy, stress tolerance, and drought resistance, which improve soil stability and moisture content (Guo et al. 2007). Cyanobacterial EPS produce binding and gluing mesh on soil particles, which apply mechanical impact on soil particles. Due to water-loving nature of EPS they retain available moisture (Mugnai et al. 2017). Similarly, inoculation of filamentous fungi enhances soil aggregation with variable potency in comparison to bacteria. The best known fungal species belong to genera of Absidia, Aspergillus, Chaetomium, Mucor, Rhizopus, Fusarium, and Aspergillus.

16.6.2 Inoculation in Plant Beneficial microbes elevate the uptake of nutrients like N, P, and K upon inoculation in crop plants. PGPR and AMF inoculation in plant is an important practice in agriculture. The crop plants secrete low molecular weight organic substances from their root passively known as root exudates, which leads to microbial community development in the root zone. The microbe in turn establishes interaction with the host plant and thereafter enhancing soil aggregation, plant growth, and escalating RAS (root adhering soil). Rashid et al. (2016) inferred that plant can absorb nutrients and water by RAS easily. Cipriano et al. (2016) summarized that B. pantoea, Rhizobium sp., and Pseudomonas sp. are well-known EPS and plant growth promoters, so they are the most investigated and best candidate for inoculation. They are directly inoculated into the seedlings. The production of EPS by microbes in the crop root zone protects the plant from several environmental stresses, also enhances the nutrient uptake in crop plants as well improves soil structure and aggregation (Alami et al. 2000; Bezzate et al. 2000). In chickpea inoculation of H. variabilis (HT-1) and Planococcus rifietoensis (RT-4) ameliorate salt stress, enhanced soil aggregation under salinity more than 75%, promoted plant growth, improved soil fertility, and under stress enhance the growth of the plant. Similarly, Rhizobium strain (YAS-34) reasonably affected water, nitrogen, and soil aggregation and RAS up to 100% under normal and water-stressed conditions. The strain enhanced the volume of soil macropore,

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increased plumule and radical biomass, and performed as a plant growth promoter. These repercussions of EPS production turned down water loss and top up water holding capacity (WHC) of soil (Alami et al. 2000). Aeromonas and Bacillus strain aggregates around the root of wheat as assessed by Ashraf et al. (2004), they observed restricted Na+ uptake in moderate saline soil. Similarly, Siddikee et al. (2010) in canola reported P. rifietoensis (RS 18) promotes plant growth, which is a halotolerant bacterium.

16.7

Amelioration of Salt Stress by Microbes

Microbial polymers produced by microorganisms are involved in salt stress tolerance, which are not only beneficial for producing microorganism but also for the associated crop. To counterbalance the harmful impact of salt stress, halotolerant bacteria form biofilm and produce EPS which have a strong affinity for water and aid in their metabolism. So, halophilic bacteria when exposed to high salt stress, it uses both strategies to vanquish the salinity stress. The Na+ ion uptake can be decreased by NaCl
tolerant strains, as they produce the polymers which can decrease the availability of Na+ ion (Upadhyay et al. 2011). Consequently, promotes survival of microorganism in saline environment by preventing osmotic stress and nutrient disparity. For example, S. meliloti (EPBI), which thrives in salty marsh at 0.3 M salt concentration, shows reduced growth when S. meliloti culture is inoculated at low concentration, for bacteria this reduced levels of salt concentration is a stressful condition (Lloret et al. 1998). Biofilm coordinates between EPS matrix and bacterial cells, i.e., bacterial biofilm has an interdependent lifestyle. It has been reported that three bacteria Halomonas aqamariana, H. meridiana, and Kushneria indalinina produce vigorous EPS and biofilm at 1 molar salinity levels. They also concluded that under saline conditions salt-loving bacteria (halophiles) colonizes crop plants root zones more effectively than normal conditions and helps plants to thrive under such stressful condition. In usara soils, halotolerant bacteria can be used as a bioremediation tool. Shah et al. (2017) quoted the potency of two halophilic bacteria Ocenobacillus kapalis and T. devorans; they produce EPS molecule which alleviates salt stress by decreasing the assimilation of Na+ ion by roots. Also, these strains ameliorate the saline soil of M. sativa (alfa-alfa) plant, as well as favored soil aggregation under salt stress and water stress equally. The glucuronic acid, glucose, mannose, and galactose are the main composition as well as the metabolic products of muran type of EPS, so they could also function as plant nutrient (Arias et al. 2003). In other legumes also a similar pattern of productivity has been reported, when chickpea (C. arientum) plant co-inoculated with the six halotolerant strains of Bacillus sp., they enhanced symbiosis effectiveness. In addition dual inoculation indeed raises the growth of plant thus suppressing the repressive effect of salt stress.

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Conclusion

Adaptation to varying environmental conditions by microorganisms especially residing in the soil through different mechanisms has well been documented after decades of studies by researchers. However, extracellular polymers secreted by microorganisms enable to maintain enough moisture around them, absorb nutrient; and also protect them from harmful organisms and stress conditions through facilitating signal-based chemical reactions. Moreover, these extracellular polymers are highly diverse owing to the difference in their composition and structure; therefore, their action is very selective. Presently, the world is moving towards the principal of sustainability where feasibility to subside the non-biodegradable and bio-incompatible compounds can be possible through the use of EPS. Gelling and emulsion capacities of EPS are contributory factors in improving soil fertility and enhancing nutrient uptake by plants. However, plant–microbial interactions are governed by the consistency and biocompatibility of extracellular polymeric substances. Characterization of EPS-producing microorganisms having different physiological potential is essential to make need-based manipulation at genetic level in these microbes to obtain improved polymers. Although, several researchers have documented that EPS ameliorates soil quality and plant growth but complex mechanism involved in the production of these compounds need to be explored extensively. Moreover, there is also a need to develop a consortium of genetically modified microorganism that can fulfill ever-increasing demand of agriculture.

16.9

Future Prospectus

Moving from green revolution to green economy in ever-increasing world population, characterization of several new EPS from microorganism is seen as the only viable solution. Hence, the use of several highly specific modern techniques to characterize new EPS and their mechanism of action need to be prioritized. Improving soil structure and fertility along with plant health management are wellestablished facts. However, implementation of techniques like next generation sequencing, CLSM, scanning electronic microscopy, nuclear magnetic resonance for characterization of EPS could open a window of its applicability in other areas too like garbage management, wastewater treatment, soil reclamation, postharvest management, etc. However, understanding of EPS structure, composition, genetic regulations under different conditions are inter-related aspects that need an in-depth knowledge base. Exploration and characterization of new polymer from microorganisms could lead to its application in other fields of scientific discoveries. Besides, studies on environment-driven signal-based EPS production and its interactions with soil and plant system are yet unexplored. Hence, it is time to integrate different fundamental knowledge together to widen the scope of better understanding and use of EPS in ever-changing climatic conditions.

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Part III Microbial Polymers in Industrial Sectors

Microbial Exopolysaccharides: Structure and Therapeutic Properties

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Hiran Kanti Santra and Debdulal Banerjee

Abstract

Microbial exopolysaccharides are the metabolic products of the bacterial or fungal cells that are composed of sugar, proteins, organic acid in various ratios and serve multiple protecting functions to the host cell against drought, pathogenic attack, antibiotics treatment, etc. They were first investigated during the 1880s and day by day have gained popularity in the sectors of food, pharmaceutics for anthropogenic benefits. They possess huge structural diversity according to their different types of sugar compositions and bond formations. Structural diversity and wide functional property have made EPSs as potent antibacterial, antioxidant, anticancer, and antitumor agents with acceptability in nano drug delivery, bioactive compound encapsulation, as prebiotics (fermented milk products like kefir, villi, taetea), etc. EPSs from LAB (lactic acid bacteria), extremophiles like marine halophiles, thermophiles, and psychrophiles are major contributors to the modern pharmaceutical industry with evidenced pharmacological applications. Their unique composition of sugars with different types of linkages is the prime cause of their multidimensional bioactivity. These exopolymers are not only useful in direct disease prevention but Also helps in therapeutic diagnosis, drug delivery, wound healing, etc. In this chapter, some recent discoveries of exopolysaccharides from a variety of microbial sources (archaebacteria, mesophiles, algal members, endophytic fungi, and bacteria) are mentioned with their utilization as antioxidative agents, antimicrobial compounds, antiproliferative compound, etc., along with their structural information.

H. K. Santra · D. Banerjee (*) Microbiology and Microbial Biotechnology Laboratory, Vidyasagar University, Midnapore, West Bengal, India e-mail: [email protected] # The Author(s), under exclusive license to Springer Nature Singapore Pte Ltd. 2021 A. Vaishnav, D. K. Choudhary (eds.), Microbial Polymers, https://doi.org/10.1007/978-981-16-0045-6_17

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Keywords

EPS · LAB · Antioxidative agents · Antimicrobial activity · Antiproliferative agents

17.1

Introduction

Exopolysaccharides from microbial sources are generally considered as polymers of carbohydrates having high molecular weight. Polysaccharides are water-soluble jelly-like thick high molecular weight long-chain polymers of sugars which have a wide range of utility in the food industry as an emulsifier, stabilizer, encapsulator, bio-thickeners and controls crystallization, syneresis. Actually, they are grouped as exopolysaccharides, capsular polysaccharides, or sometimes lipopolysaccharides. It is very common that capsular polysaccharides are responsible for the pathogenicity of the virulent strains whereas lipopolysaccharides are generally stored in the outer membrane of the bacterial cell. EPSs are composed of carbohydrate and non-carbohydrate parts (like-acetate, phosphate, succinate, pyruvate, etc.). According to the monosaccharide compositions they are of two types—homo and heteropolysaccharides which are made up of the same (pullulan, cellulose, dextran, etc.) or different (hyaluronic acid, xanthan) type of repeating monosaccharide units. Due to their viscous pseudoplastic behavior, they are popular emulsifying, stabilizing, and viscosifying agents that retain moisture in food, improves blood flow, acts as a microcarrier in tissue culture and matrix immobilizing agents. Their intense and diverse biological function is obtained due to their composition, structure, and molecular weight. Polysaccharides are known to be present in different type of life forms ranging from prokaryotic to eukaryotic ones and display multidimensional biochemical structures and physiochemical qualities. They are necessary biological natural products reported from microorganisms, animals, and plants with popular biological potentials, e.g., rheology modifiers in food and pharmaceutical industries (as antimicrobial—antibacterial, antifungal, antiviral, antitumor, antioxidant, anticancer, cholesterol lowering, prebiotics, etc.), cosmetics, and textile industries. They offer the host cell adhesion and protection against harsh environmental situations. Microbial EPSs are characterized by short production times and simple extraction procedures. Microbial EPS are of high demand than plant or based on their physiochemical (e.g., freezing, boiling, and also melting) characteristics and rheological (characters related to flow of the viscous liquid) characteristics. They are manufactured at logarithmic (exponential or log) or stationary (stable, i.e., birth of new microorgnisms ¼ death of old organisms) are found in insoluble or soluble way in the culture extracts as slime components. Their monomeric (simplest and smallest) units, glycosidic linkages (covalent bond that joins sugar) and diverse branching pattern has made them unique. Microscopic fungi derived EPS are of higher demand and easy to handle than the fruiting body derived EPS due to their easy isolation, simple purification, minimum cost, and gigantic production. The optimum conditions include the utilization of submerged culture where the dispersed

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mycelial filaments produce interwoven hyphal pellets excreting polymeric substances. Plant (alginate, carrageenan, guar gum, locust bean gum, pectin, and starch) and animal (casein, gelatin) polysaccharides after certain modifications are widely used as hydrocolloids but their use is restricted due to limitations of their rheological properties and stability. So the focus is shifted towards the microbial source. Microbial (microalgal, bacterial, fungal) extracellular polysaccharides or exopolysaccharides remains associated with cellular surfaces forming a capsular or slimy appearance and are sometimes involved in immune response (capsular EPS and O-antigen in case of Streptococcus pneumoniae and S. agalactiae). Microbial cells use their EPS as a shield of protection against antibiotics treatment, disinfecting chemicals like ethanol, sulfur dioxide, metal ions, desiccation, phagocytosis, predation by protozoan, osmotic stress, and macrophage attack. It promotes biofilm formation, adhesion to solid substratum, and facilitates cellular recognition. Popular microbial biopolymers include bacterial alginates, pullulan, dextrans, yeast glycans, xanthan, and gellan. The prime hindrance with microbial polysaccharide is the detailed knowledge of the biosynthetic pathway, genetic regulations, expensive recovery costs, and critical bioprocess formulations. But once the issues are resolved microbial polysaccharides positively influence the food and pharmaceutical sectors. Another major drawback is the safety issues related to no GRAS (generally recognized as safe) organisms like Xanthan and gellan from phytopathogenic bacterial strains of Xanthomonas campestris and Sphingomonas elodea, respectively. In this respect, principle sources of exopolysaccharides are food-grade microorganisms, GRAS-LAB, etc.

17.2

LAB Polysaccharides

Microbial polysaccharides not only function as a direct therapeutic agent but also acts as a prebiotic that promote the growth of beneficial microbiomes in intestinal tracts and suppress the flora of putrefying or gas-forming bacteria. Lactic acid bacteria also known as LAB mainly isolated from dairy products like yogurts, kefirs, fermented milks, cheese, etc., are potent producers of exopolysaccharides with multidimensional application (Fig. 17.1). Other than LAB Propionibacteria, Bifidobacteria are also EPS producers. The popular fermented milks of Scandinavian origin like villi, taetea, skyr are of high demand due to their slimy to thick consistency because of the souring effect of Lactobacillus lactis subsp. lactis and L. lactis subsp. cremoris and texture of heteropolysaccharide. Other than these drinks kefir, a type of alcoholic to acidic effervescent drink fermented from grains is also acceptable due to its exopolysaccharide contents of the LAB strains (Vuyst and Degeest 1999). LAB, when fall under GRAS category, can provide a better natural end product in terms of enhanced and improved texture with rheological property, low viscosity, increased moisture content, and higher water retention capacity. Major drawbacks of LAB exopolysaccharides are in vivo biofilm formation and bio-fouling leading to loss of palatability of milk products, spoilage of wines, beers, ciders,

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Fig. 17.1 Common fermented food with LAB exopolysaccharides and their potential multidiscipline therapeutic applications

fermented sausage and airtight cooked meats, etc. Dextran derivatives, levan, and alternan from LAB are used in the paper and metal plating industry, develop products that improve blood flow and extend blood volume, stabilize food syrup as a thickener, acts as a bulking agent with low viscosity, acts as an extender in cosmetics and food industry, etc. In the year of 1969 US FDA (United States FOOD and Drug administration authority) first legally approved the commercial use and application of bacterial exopolysaccharide (xanthan from Xanthomonas campestris) as a food additive for thickening, gelling of sugar beet and cane syrups. Hydrated polymers of xanthan are popularly used in soups, sauces, yogurts, dairy desserts, salad dressings, and fermented milk products due to its thixotropic and shear thinning (pseudoplastic) properties. Its rheology falls rapidly upon constant pouring, shaking, and stirring but regains its nature immediately after the shaking is withdrawn. Its wide range of adaptability over pH changes, different ionic strengths, and temperature has made it more compatible with other food products. The negative relationship between shearing force and retention of viscosity is highly recommended for palatability, i.e., mouth fullness texture. Slime mold producing bacterial cultures are coupled with the starter LAB cultures as they improve product viscosity, lower syneresis problems, and avoid graininess issues to some extent. These ropy strains maintain the regularity of stirred type yogurt as the viscous EPS is less susceptible to mechanical damage done by blending and pumping procedures making the coagulum more thermal and physical shock resistant. Another positive side of this type of approach is that it does not require any artificial stabilizers making the product much more natural and acceptable. The higher acceptability of fermented milk products are

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due to their viscosity that is enhanced by the inoculating bacterial strains changing the rheology making the products more popular. The viscous nature depends on the pH, fermentation time, culture medium, culture strains, and also total solids of the medium. Not only the use of ropy, moderately ropy, or non-ropy strains affects the texture of the product but also the aggregated proteins influence the taste parameters. The ropy (Lactobacillus delbrueckii subsp. bulgaricus strain) and non-ropy both types of starter cultures are potent enough to form a uniform layer of EPS strands in between the microbial cells and casein protein networks modifying the protein texture and aggregation properties. Yogurts are made up of stabilizers; either commercially available chemicals or derived from the ropy starter culture of some particular LAB. Yogurts with natural stabilizers from microorganisms’ source have distinct rheology and palatability than the artificial ones. Another popular milk product with increased adhesiveness due to the action of polysaccharide producing LAB strains (L. lactis subsp. cremoris) is known as Villi. It prepares a thick slimy matrix where the proteins are trapped forming the casein micelle cluster increasing the consistency of the product. The main two constituents of the kefir grains are LAB (Lactobacillus kefiranofaciens) and yeasts (Candida kefir) which prepare the polysaccharide gel-like matrix (kefiran). The homo and heterofermentative strains are Lactobacillus acidophilus, Lactobacillus kefirgranum, and L. kefir and L. parakefir. Panthavee et al. (2017) isolated two EPS producing strains of thermophilic LAB (LY45-Pediococcus pentosaceus, PY45-Lactobacillus amylovorus) from Thai tropical fruits. The strains (LY45 and PY45) produced neutral to acidic polysaccharides and manufactured glucose (C6H12O6), mannose (D-mannose), galactose, respectively. The EPS shows anti-hyaluronidase activity in a similar resolution with sodium cromoglicate and dipotassium glycr-rhizinate. So these types of strain are very much useful in pharmaceutical product development that can act as both probiotics and anti-allergic or anti-inflammatory. Luang-In et al. (2018) determined the bioactivities of EPS isolated from Bacillus tequilensis PS21 obtained from kefir of Kampaeng Petch, Thailand. FTIR, HPLC, and SEM data confirmed the heteropolysaccharide (monosaccharide components are xylose, glucose, ribose, rhamnose, galactose with 17.65, 2.54, 1.83, 1.23, 1%, respectively) nature of the protein-bound polysaccharide with a grainy appearance and uneven porous surface. The antioxidant nature of the EPS was confirmed by DPPH, ABTS, and FRAP methods. It is also characterized by anti-tyrosinase activity with 34.9% inhibition. This type of study emphasizes the multidimensional role of EPS not only as prebiotics but also as a source of antioxidants. Yu et al. (2018) screened a new LAB (Weissella cibaria 27) from kimchi, a Korean dish and documented the antibacterial activity of the EPS produced by this strain under optimum conditions (22  C, pH 62, 24 h). EPS is made up of dextran with α-1,6 glycosidic linkages having a molecular weight of 1.2  107Da. The use of sucrose has changed the surface property of EPS and made it more hydrophobic for better inhibition of bacterial pathogens. Thus, these types of EPS possess potent application in the sector of food, pharmaceuticals, cosmetics industry, and development of chemotherapeutic agents with antibacterial activity.

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EPSs from Marine Microbial Sources

In most of the cases, EPSs are of low poisonous activity and this property has made them being widely used as a therapeutic agent. EPSs suppress the tumor development by their immunomodulatory activity in vivo. They exhibit toxicity to malignant tumor cells by boosting up the host’s defense and resistance. Marine microorganisms also play an important role in the production of exopolysaccharides with therapeutic properties. Marinactan is an exopolysaccharide isolated from marine Flavobacterium uliginosum (an isolate of surface seaweed) a heteroglycan made up of glucose, mannose, and fucose monomer shows anticancer activity towards solid and ascites form of sarcoma 180 form (Umezawa et al. 1983). In another study conducted by Ruiz-Ruiz et al. (2011) they screened the two EPS producing strain for antitumor activity and reported that the heteropolysaccharides restrict the proliferation of tumor infected cell lines and oversulfated ones are effective against T-cell lines (obtained from acute lymphoblastic (blood and bone marrow cancer) leukemia-intrinsic pathway). Chen et al. 2011 isolated Aspergillus sp. Y16 from the leaves of mangrove plant Ipomoea pescaprae (Linn.) and extracted, purified a homogeneous exopolysaccharide (As1-1) by AEC (anionexchange) and GPC (gel permeation chromatography). 1D and 2D NMR spectroscopical data suggested As1-1 was made up of mannose (molecular weight15 kDa). Backbone of the polysaccharide is made up of (1-2)-linked α-Dmannopyranose units, substituted at C-6 by the (1-6)-linked α-D-mannopyranose, (1-)-linked β-D-galactofuranose and (1-)-linked β-D-mannopyranose units. The polysaccharide was characterized by in vitro antioxidative potency tested using DPPH (1,1-diphenyl-2-picrylhydrazyl), superoxide radicals, and in vivo affectivity was confirmed using lipid peroxidation and superoxide radicals. Ye et al. (2012) isolated and purified an acidic exopolysaccharide from a marine Pseudomonas sp. PF-6 which was water-soluble but insoluble or immiscible in organic (acetone and benzene, etc.) solvents and odorless with a (MW) molecular weight of 8.83  105 Da. After oxidization, it turns to uronic acid and IR spectroscopic analysis confirmed the absorption bands at 927.49 cm1 and 811.76 cm1 which elucidate the occurrence of the pyran group. The EPS possess antioxidative potentiality confirmed through DPPH, OH radical scavenging activity. The antioxidative nature was directly proportional to the concentration of EPS. Ye et al. (2016) isolated a selenium tolerant strain of Pseudomonas sp. PT-8 from Antarctic Ocean mud and characterized the selenium-rich polysaccharide (Se-EPS) with high uronic acid content. EPS exhibited a yield of maximum 256.7 mg/kg of selenium upon optimization. EPS was purified into two fractions; one being neutral and another being acidic where the second acidic fraction (EPS2) being a homogenous polysaccharide with rhamnose (C6H12O5)19.58:arabinose (C5H10O5) 19.28: xylose (C5H10O5) 5.97: mannose (C6H12O6) 18.99:glucose (C6H12O6) 23.70:galactose (C6H12O6) 12.48 with 7.3 kDa MW (molecular weight) having a molecular weight of 7.3 kDa. The amount of rhamnose and the molecular weight of the EPS is involved in a negative relationship. This selenium-rich EPS is a good agent of antioxidative activity confirmed through DPPH and OH radical scavenging assay.

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Another example includes YCP which is a (1,4)-α-D-glucan obtained Phoma herbarum S4108 (a filamentous fungi found in sediments) can influence the activation of macrophage (2). Two different types of neutral exopolysaccharides (GX1-1, GX2-1) were reported from an indigenous fungus Penicillium sp. gxwz446 isolated from a coral Echinogorgia flora occurring in South China region (Sun et al. 2016). EPS were purified by AE (anion-exchange), GPC (Gel Permeation chromatography). Spectroscopic analysis coupled with chemical testing revealed the nature of the EPS as glucan with a molecular weight of 5.0 kDa. GX1-1 has (1 ! 4)-linked α-Dglucopyranose units as the backbone with a substitution at C-2 by a single α-Dglucopyranose on every sixth sugar residues. GX2-1 was made up of galactofuranose with mannogalactoglucan with a molecular weight of 9.5 kDa. The prime linkages consists of (1 ! 4)-β-D-Glcp, (1 ! 5)-β-D-Galf, (1 ! 3,5)-β-D-Galf, (1 ! 6)-α-D-Manp and (1 ! 2, 6)-α-D-Manp. GX1-1 can cause macrophage activation. GX1-1 was subjected to sulfated modification and there was one sulfate substitution on every sugar ring basically at O-6. The sulfated derivatives were more efficient to enhance the pinocytic ability of RAW264.7 cells and also promote NO production. It is not always that exopolysaccharides derived from microorganisms directly contribute to therapeutic purposes but sometimes in a passive way they modify the production of some bioactive component in another eukaryotic host such as plant cells (Chen et al. 2016). One such example includes the higher production of volatile oil components of Atractylodes lancea due to the elicitation of exopolysaccharide components produced by the endophytic fungus Gilmaniella sp. AL12 (a stem isolate of A. lancea). Researchers identified that the active component is an extracellular mannan having a polymerization degree of 26–42. Results of 2D electrophoresis membrane proteomics study revealed that 83 different proteins are expressed and probably mannan maintains an antagonistic between endophyte AL12 and host A. lancea interactions. Mannose was produced from mannan that mediates activation of enzyme hexokinase and also promotes anabolic biochemical reaction photosynthesis promoting photosynthesis coupled with the metabolism of energy (breakdown of product for the generation of energy-ATP) that mediates important metabolic fluxes (turnover of components into something new) for terpenoid biosynthesis. The remnant part of the mannan enhances autoimmunity of the host plant by Growth protein-mediated signal transfer coupled with pathways that bind mannose and lectin. As a result of the immune-stimulating activity, the higher accumulation of volatile components occur. The prime constituents of the A. lancea volatile oil are sesquiterpenes and polyacetylene that includes atractylone, atractylodin, β– eudesmol, b-caryophyllene, caryophyllene oxide, hinesol, b-sesquiphellandrene, and zingiberene (Yuan et al. 2016). The plant constituents were used as basic raw or crude drug for the treatment of rheumatic diseases (problems of tendon and ligament, etc.), gastric problems, and also as diuretics in Asian countries like China, Japan, and Korea (Ouyang et al. 2012). Other pharmacological utilities of this herb include anti-inflammatory (prime component for mast cell-mediated inflammatory diseases), antimicrobial, anticancer, antihypertensive, lipase inhibition, anti-obesity, antiplatelet, anti-ulcer, antipyretic, etc. (Jiao et al. 2014).

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Yan et al. (2016) evaluated the antioxidative potentiality of an EPS obtained from Aspergillus versicolor N2bc which was mannoglucogalactan (molecular weight20.5 kDa) in nature. The main chain of EPS consists of !2)-D-Glcp-(1!, !2)-DGlcp-(1! and !6)-D-Manp-(1 ! units, substituted at C-6 position of !2)-D-Glcp(1 ! units. Branches were made up of galactofuranose (five-membered ring form of galactose)-oligosaccharides (made up of a small number of monosaccharide units) built up of !5)-D-Galf-(1!, !6)-D-Galf-(1 ! and terminal -D-Galf units). El-Newary et al. (2017) evaluated the antitumor, antioxidant, and antiinflammatory (COX-1 and COX-2) activity of the EPS which is acidic in nature and composed of monosaccharide units of glucose, galactose, glucuronic acid (molar ratio of 1.6:1.0:0.9) having a molecular mass of 3.76  104 g/mol. Except reducing power all the other common measurement units of antioxidative nature like free radical scavenging, antiproliferative activity towards cancer cells (breast cancer) MCF7 exhibited IC50 and IC90 values of 70 mg/mL and 127.40 mg/mL, respectively. The antitumor activity of the EPS was due to uronic acid contents that may find potential application in the food and pharmaceutical industry. Hassan and Ibrahim (2017) investigated the antitumor (effective against MCF-7, HCT-116, and HepG2), antibacterial against Aeromonas hydrophila (Gramnegative), Pseudomonas aeruginosa (Gram-negative), Streptococcus faecalis (Gram-positive) effect of heteropolysaccharide obtained from marine bacterial species Bacillus subtilis SH1 which was composed of monosaccharide units of maltose and rhamnose having a compact film-like structure. The production parameters were optimized 2.5% peptone-protein source, 4.5% yeast extract-nitrogen source, and malt extract-sugar source to enhance the yield up to 1.4 times. It was also characterized by antiviral activity along with antioxidant potency at higher concentrations. Due to its film-like appearance, it could be beneficial for the preparation of plasticized films. Abdrabo et al. (2018) isolated 13 marine bacterial species from algal and rock surfaces of Suez Gulf, Egypt and detected the antimicrobial and antitumor effect of the biofilm-associated exopolysaccharide. Halomonas saccharevitans AB2 was the most potent isolate with antimicrobial (antibacterial and antifungal against Vibrio fluvialis and Aspergillus niger ATCC 16404, respectively) and antitumor activity (against eight cell lines) with an IC50 value of 20.3 μg mL against A-549 (human lung carcinoma) making this strain a therapeutically valuable EPS reserve. Mohamed et al. (2018a, b) screened 29 bacterial strains or the production of exopolysaccharide from soil sediments of mangrove area of Ras Mohamed area, Read Sea Cost of Sinai Peninsula, Egypt. One strain (identified as Bacillus altitudinis MSH2014) provided a satisfactory yield of EPS and monosaccharide components were mannuronic acid, glucose, sulfate, and molecular weight is 4.23  105. EPS shows potent anticancer activity against two cancer cell lines EACC and lung cancer A-549. Exopolysaccharides from marine microorganisms are known to be effective against liver cancer (malignant) cell lines also (Yang et al. 2013, 2014; Peng et al. 2014). Eleven bacterial strains were obtained from saltwater marine sediments of the Mediterranean and the Red Sea and they were tested for their cytotoxic nature

17

Microbial Exopolysaccharides: Structure and Therapeutic Properties

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against HepG2 cells. It was reported that five strains (Bacillus amyloliquifaciens MGA2, Bacillus thuringiensis E4, Brevundimonas subvibrioides MSA1, Pseudomonas fluorescens SGA3 (Gram-negative), Advanella kashmirensis NRC-7 (Gramnegative)) were the most effective against HepG2. These are acidic heteropolysaccharides (made up of different units of monosaccharides) with hexose sugar glucose, galactose, mannuronic acid, mannose, glucuronic acid as monosaccharide units. The occurrence of sulfate and carboxylic groups in different amounts (percentages) were obtained from FTIR data. The molecular weight ranged from 1.94  104 to 7.95  105 g/mol in an average. (Asker et al. 2018). Abinaya et al. (2018) documented the antibiofilm (biofilm inhibitory) and antilarval functions of exopolysaccharide obtained from a marine Bacillus licheniformis Dahb1 which was composed of aromatic compounds and various functional groups. EPS exhibited strong antioxidative property based on DPPH (2, 3, 5 diphenyl picryl hydrazine) free radical scavenging ability. The antibiofilm activity was against Gram-negative bacteria—Pseudomonas aeruginosa and Proteus vulgaris; Gram-positive bacteria—Bacillus subtilis and Bacillus pumilus; Candida albicans at 75 μg/mL concentrations. Antilarval activity was against Anopheles stephensi and Aedes aegypti with an LC50 value of